CellutionsVol 2: 2012The Newsletter for

Cell Biology Researchers

EMD Millipore is a division of Merck KGaA, Darmstadt, Germany

What killed your cells?

Multiplexed detection of changing cytokine profiles with respect to PBMC stimulation, sepsis and autoimmune diseases Page 7

LentiBrite™ Lentiviral Biosensors for fluorescent cellular imaging: analysis of the cytoskeleton Page 11

Cellular characterization of chromatin state via high throughput ChIP assay Page 16

REVIEW – Sonic Hedgehog: its dual role in morphogenic and mitogenic signaling Page 21

To subscribe to the quarterly Cellutions newsletter,please visit www.millipore.com/cellquarterlynews

Simplified evaluation of apoptosis using the Muse™ Cell Analyzer Page 3

2

Product HIGHLIGHt

Now, experience it. Smarter cell analysis with your own Muse™ Cell Analyzer.Revolutionize the way you analyze cell viability, apoptosis and cell cycle with the Muse™ Cell Analyzer. Using miniaturized fluorescence-based detection, a user-friendly interface and optimized assays, the Muse™ Cell Analyzer provides powerful cell analysis simply, accessibly, and affordably. Experience the new Muse™ Cell Analyzer and make smarter, faster and more accurate decisions about your experiments.

www.millipore.com/muse

Simply see more.Scan this 2d bar code with your mobile device.

3

IntroductionThe degree of apoptosis in a cell population is an important

parameter that contributes to a comprehensive picture

of cell health. Accurate detection and measurement of

cellular apoptosis is essential for drug development and

discovery, for understanding mode of compound action,

and for understanding the impact of culture and growth

conditions. However, the assessment of cellular apoptosis

has been limited due to the requirements for expensive

and complicated instrument platforms, expertise and

improved analytical methods that provide rapid, robust and

reproducible apoptosis data. Access to these improvements

can facilitate apoptosis monitoring, thereby enabling the

efficient, daily execution of cellular research.

The Muse™ Cell Analyzer is a novel instrument that enables

multidimensional cell health analysis on a single platform.

This small, benchtop cell analyzer effortlessly guides users

through the acquisition and analysis of samples using

a highly simplified and intuitive touchscreen interface

which delivers rapid measurements of cell concentration,

viability, apoptotic status, and cell cycle distribution1. Using

multiparametric fluorescent detection of individual cells

via microcapillary flow technology, the system enables

highly sensitive and rapid detection of cellular samples

using minimal cell numbers. The simplified format enables

researchers of varying backgrounds and experience levels

to obtain a comprehensive picture of cellular health.

In this study, we used the Muse™ Cell Analyzer for

apoptosis studies using the Muse™ Annexin V & Dead

Cell Assay, a rapid, no-wash assay for the identification

of apoptotic cells. Jurkat and HeLa cells were treated with

a range of cytotoxic and anti-tumor compounds, and the

results from percentage of live, early, late apoptotic, and

dead cells were evaluated. Our results demonstrate that

the assay provides rapid and sensitive detection of cellular

apoptosis and provides quantitative and precise results on

a variety of cellular types and compound treatments.

Materials and MethodsAssay principleApoptosis, or programmed cell death, is an important

regulator of cell growth and proliferation and is

characterized by distinct morphological changes. One

key early aspect of apoptosis is the translocation of

phosphotidylserine from the inner to the outer leaflet of

the plasma membrane and exposure to outer surface of the

cell. This universal phenomenon is independent of species,

cell type and induction system and occurs early in the

apoptotic process.

The Muse™ Annexin V & Dead Cell Assay is based on the

detection of phosphatidylserine (PS) on the surface of

apoptotic cells and uses a premixed reagent containing

fluorescently labeled Annexin V in combination with a

dead cell marker, 7-AAD. Annexin V is a Ca2+-dependent

phospholipid-binding protein that has a high affinity for

PS, a membrane component normally localized to the

internal face of the cell membrane. Early in the apoptotic

pathway, molecules of PS are translocated to the outer

surface of the cell membrane where Annexin V can readily

bind to them (Figure 1). Late-stage apoptotic cells show

loss of membrane integrity. The membrane-impermeant

dye, 7-AAD, is used to distinguish dead cells from early

apoptotic cells. The assay can thus distinguish four

populations:

• Viable cells, not undergoing detectable apoptosis:

Annexin V (–) and dead cell marker (–)

• Early apoptotic and dead cells: Annexin V (+) and dead

cell marker (–)

• Late apoptotic or dead cells: Annexin V (+) and dead cell

marker (+)

• Cells that have died through non-apoptotic pathway:

Annexin V (–) and dead cell marker (+)

Simplified evaluation of apoptosis using the Muse™ Cell AnalyzerAsima Khan, Katherine Gillis, Julie clor, Kamala tyagarajanEMD Millipore Corporation

Figure 1. The combined use of fluorescent labeled Annexin V and membrane-impermeant 7-AAD DNA-binding dye can distinguish live, early apoptotic, and late apoptotic cells.

Healthy Cell Apoptotic Cell Late Apoptotic Cell

4

Sample preparationChinese Hamster Ovary (CHO), HeLa, and Jurkat cells were

kept in log phase growth in complete medium.

Prior to assaying, cells were treated with compounds as

indicated in Figures 4-9. The assay employs a simple mix-

and-read procedure, as shown in Figure 2. 100 µL of cells

was mixed with 100 µL of Muse™ Annexin V & Dead Cell

Assay Reagent in 1.5 mL screw-cap microfuge tubes and

incubated for 20 min in the dark at room temperature.

Data from prepared samples were quickly acquired using

the step-by-step instructions on the touchscreen and

the Muse™ Annexin V & Dead Cell Software Module as

shown in Figure 3. The assay provides results on counts

and concentrations of the four cell populations described

above.

Key features of the assay include:

• Mix-and-read assay minimizes loss of fragile,

apoptotic cells

• Highly simplified acquisition and analysis, guided

through touchscreen interface

• Accurate and precise data

• Minimal number of cells required

• Validated with both suspension and adherent cell lines

data AcquisitionData from prepared samples were quickly acquired using

the step-by-step instructions on the touchscreen and the

Muse™ Annexin V & Dead Cell Software Module as shown

in Figure 3. Briefly, a user enters the Annexin V & Dead Cell

Module and hits “Run Assay”. The touchscreen prompts

the user to load a sample and, through simple on-screen

instructions, guides the user through the optimization

and verification of settings. The user then enters sample-

specific information and then touches “Run Sample.” The

instrument displays the results screen with the calculated

concentrations of live, early apoptotic, late apoptotic, and

dead cells. The instrument displays the results screen with

the values and provides the user the option to view the

dotplot as well as adjust markers between samples.

result parameters include information on:• Population percentages and concentrations

• Total cells per mL

• Dilution factor (input value)

• Sample number

• Sample ID

Data can be stored on the device, exported in a report

format and/or exported as a Microsoft Excel® file, enabling

the production of a robust documentation trail with

experimental details preserved.

ADD CELLSPREPARE CELLS ADD REAGENT READ RESULTS

Culture cells toinduce apoptosis

Add 100 µLcells

Add 100 µLreagent

Mix, incubate atRT for 20 minutes

Figure 2. Workflow for Muse™ Annexin V & Dead Cell Assay. The assay utilizes a simple mix-and-read procedure and provides quantitative apoptosis data.

Figure 3. Steps to perform acquisition and analysis using a guided user interface and software module. results on apoptotic cell percentages and concentrations are displayed automatically at the completion of acquisition. optional dotplots allow for visualization and further data manipulation.

open & Load Sample Adjust Settings Acquire results

5

resultscomparison of accuracy with alternate methods of apoptosis measurementThe accuracy of test results obtained on the Muse™ Cell

Analyzer was compared with traditional methods for

apoptosis analysis. Adherent CHO-K1 cells were treated

with 0.25 µM staurosporine for 16 h and suspension Jurkat

cells were treated with 1 µM staurosporine for 4 h. Samples

were stained in triplicate following the manufacturer’s

instructions and analyzed using either Muse™ Annexin V

& Dead Cell Assay, fluorescent microscopy, image-based

fluorescent analysis, or the guava® Personal Cell Analyzer

(PCA). The results for the percent of apoptotic and dead

cells clearly indicate that the Muse™ Cell Analyzer provides

equivalent cell population measurement results, compared

to results from other predicate analysis methods, for both

adherent and suspension cell lines (Figure 4).

Figure 4. Apoptosis measurements are consistent with other cell analysis methods. cHo-K1 and Jurkat cells were treated with staurosporine to induce apoptosis. the data show the comparison of population percentages for all 4 methods (Fluorescent Microscope, Image-Based Automated device, Personal cell Analyzer, and Muse™ cell Analyzer). Each point represents the average of three samplings.

Figure 5. Superior precision for apoptotic percentage detection, compared to other analysis methods. data are based on triplicate measurements of 10 samples from suspension and adherent cell lines treated with staurosporine to induce apoptosis.

Figure 6. Apoptotic impacts of multiple compounds on Jurkat suspension cell line. untreated Jurkat cells (Top, left) were compared with cells treated for 4 h with 10 µM camptothecin, a DNA topoisomerase inhibitor (Top, right), 4.7 µM gambogic acid, a compound with potent anti-tumor activity (Bottom, left), or 150 µM diamide a thiol-oxidizing agent (Bottom, right) using the Muse™ Annexin V & Dead Cell Assay.

Evaluation of precision and reproducibility To assess precision, triplicates of 10 samples from adherent

and suspension cell lines treated with staurosporine to

induce apoptosis were prepared and analyzed them using

Annexin V-based assays using the fluorescent microscope,

an image-based fluorescent device, or the Muse™ Cell

Analyzer. Average coefficients of variation (%CVs) for the

early and late apoptotic populations were calculated for all

devices and compared. The average %CVs obtained with the

Muse™ Annexin V & Dead Cell Assay were consistently lower

than those seen using other comparative methods for the

same samples (Figure 5).

Studying Apoptotic Impacts of compounds on Multiple cell typesThe apoptotic effects of several compounds were evaluated

using Jurkat cells (suspension) and HeLa cells (adherent).

The results in Figure 6 demonstrate that the differential

impacts on Jurkat cell health caused by the compounds

could be discriminated by the assay. Camptothecin

treatment caused early apoptosis with relatively little cell

death, gambogic acid treatment caused early apoptosis as

well as low levels of cell death, and diamide resulted in the

appearance of predominantly late apoptotic/dead cells.

Results from the analysis of the adherent HeLa cell line

are shown in Figure 7. Again, the assay enabled clear

discrimination of apoptotic and dead cell populations.

Both anisomycin and diamide resulted in high percentages

of late apoptotic or dead cells and very little early

apoptosis, while camptothecin treatment resulted in the

appearance of equal proportions of early and late apoptotic

populations.

Live Apoptotic

CHO-K1

Apoptotic

Jurkat

Apoptoticand dead

20

10

30

40

Perc

ent C

ell P

opul

atio

n 50

60

0Apoptoticand dead

Live

Fluorescent microscopyImage-based automated device

PCAMuse™ Cell Analyzer

4

8

12

16

FluorescentMicroscope

FluorescentImage-based

Device

% C

V

0Muse™ Cell

Analyzer

A. untreated

c. Gambogic acid

B. camptothecin

d. diamide

6

references1. Gillis, K, et al. Precise and Accurate Counts and Viability

Measurements Across Multiple Cell Lines Using the Muse™ Cell Count & Viability Assay. EMD Millipore Cellutions Newsletter. 2011; Vol. 4:p.3-7.

2. Guizzunti, G. Subcellular Localization and Activity of Gambogic Acid. Chembiochem. 2012 Apr 24.

description catalogue No.

Muse™ Cell Analyzer 0500-3115

Muse™ Count & Viability Assay MCH100102

Muse™ Annexin V & Dead Cell Assay MCH100105

Muse™ Cell Cycle Assay MCH100106

Muse™ Caspase 3/7 Assay MCH100108

Muse™ MultiCaspase Assay MCH100109

Muse™ MitoPotential Assay* MCH100110

*Available July 30, 2012.

Available from www.millipore.com.

RELATED pRoDUCTS

Figure 7. Analysis of apoptosis and cell death in HeLa cells induced by various compounds. Untreated HeLa cells (Top, left) were compared with cells treated for 16 h with 100 µM anisomycin, an inhibitor of DNA synthesis (Top, right), 20 µM camptothecin, an inhibitor of the DNA enzyme topoisomerase I (Bottom, left), or 150 µM diamide, a thiol-oxidizing agent (Bottom, right) using the Muse™ Annexin V & Dead Cell Assay.

Figure 8. dose response data obtained for Jurkat cells treated with staurosporine for 4 h. Jurkat cells were treated with multiple concentrations of staurosporine for 4 h then analyzed using the Muse™ Annexin V & Dead Cell Assay. Each data point represents a triplicate sampling at each concentration.

Figure 9. dose response data obtained for Jurkat cells treated with gambogic acid. Jurkat cells were treated with multiple concentrations of gambogic acid for 4 h then analyzed using the Muse™ Annexin V & Dead Cell Assay. Each data point represents a triplicate sampling at each concentration. Error bars represent standard deviation.

dose response studies with multiple inducersSimplified dose response measurements are important to

understanding mode of compound action. Jurkat cells were

treated with multiple concentrations of staurosporine,

a known protein kinase inhibitor, for 4 h then analyzed

samples using the Muse™ Annexin V & Dead Cell Assay

(Figure 8). At all concentrations, staurosporine treatment

caused cells to primarily undergo early apoptosis, exhibiting

little or no death. On the other hand, gambogic acid, a

compound with potent anti-tumor activity, caused rapid

apoptosis and cell death at relatively low concentrations

(Figure 9). At concentrations of gambogic acid below

18.75 µM, a higher proportion of early apoptotic cells were

observed, while increasing concentrations resulted in a

predominance of late apoptotic/dead cells, even given the

short duration of treatment. These results are consistent

with the established classification of gambogic acid as a

potent inducer of apoptosis2.

conclusionsThe Muse™ Cell Analyzer provides a simple, powerful

method for obtaining important apoptosis information.

Our results indicate that the mix-and-read Muse™ Annexin

V & Dead Cell Assay and software module enabled the

acquisition of accurate and highly precise measurements

of cellular apoptosis. The assay is versatile and works

with both suspension and adherent cell lines and multiple

treatment conditions. The Muse™ Annexin V & Dead Cell

Assay has the potential to greatly simplify the study of

apoptosis and enable researchers to easily obtain dose-

based and mechanistic information in the drug discovery

process for compounds of interest.

40

20

60

80

100

% o

f Po

pula

tion

00.0001 0.001 0.01 0.1 1.0 10

4.691 9.38 18.75 37.5 15075

102030405060708090

100

0

µM Inducer

% O

f Pop

ulat

ion

LiveEarly ApoptoticLate Apoptotic/Dead

4.691 9.38 18.75 37.5 15075

102030405060708090

100

0

µM Inducer

% O

f Pop

ulat

ion

LiveEarly ApoptoticLate Apoptotic/Dead

A. untreated

c. camptothecin

B. Anisomycin

d. diamide

7

IntroductionTh17 cells are recently discovered T helper (Th) cells

that play important roles in the establishment and

maximization of the capabilities of the immune system1.

Specifically, activated Th17 cells secrete the cytokines

IL-17A, IL-17F, IL-21, TNFα, and IL-22 to promote tissue

inflammation (Figure 1).

Th17 cells are involved in the clearance of extracellular

bacteria and fungi. They are abundant in the intestinal

lamina propria and function as a barrier against invading

pathogens2-4. Excessive amounts of Th17 cells have been

implicated in the pathogenesis of several autoimmune

diseases, including multiple sclerosis, psoriasis, juvenile

diabetes, rheumatoid arthritis, Crohn’s disease and

autoimmune uveitis1. In addition, Th17 cells play an

important role in tumor development, progression and

metastasis, potentially in both promoting and inhibiting

tumor growth5.

Because Th17 cells, like most immune cells, deploy a

multi-cytokine response to stimulants, understanding their

function efficiently, while conserving precious samples,

requires simultaneous measurement of these multiple

analytes.

We recently developed MILLIPLEX® map Human and Mouse

Th17 multiplex assays for simultaneous measurement

of the cytokines that are secreted from Th17 cells and/

or regulate Th17 cell differentiation and activation. In

this study, we used the two Th17 panels to analyze the

cytokine secretion in human and mouse PBMCs stimulated

with lipopolysaccharide (LPS), concanavalin A (Con A) or

phytohemagglutinin (PHA).

LPS, a component of Gram-negative bacterial cell walls,

stimulates the innate immune response via TLR (Toll-like

receptor) 4. Con A and PHA are lectins, which bind to cell

membrane carbohydrates and induce cell agglutination,

T-cell mitosis and differentiation.

We also examined cytokine levels in human plasma from

sepsis, rheumatoid arthritis (RA) and lupus patients and

examined the cytokine response in mouse plasma at

various time points after in vivo LPS-challenge.

Multiplexed detection of changing cytokine profiles with respect to pBMC stimulation, sepsis and autoimmune diseasesrobert Keith, Munmun Banerjee, tim Warmke, and Qiang XiaoEMD Millipore Corporation

Figure 1. th17 cells arise as an independent lineage. Based on numerous studies in mouse systems, Th17 cells are produced from T helper progenitor (Thp) cells in the context of IL-6 and TGFβ. IFNg and IL-4 inhibit Th17 differentiation, while simultaneously promoting differentiation of Th1 and Th2 cells, respectively. Treg cells develop in the presence of TGFβ stimulation in the absence of an inflammatory signal such as IL-6.

Cell-mediated Immunity

IFNγLT

IL-17AIL-17FIL-6TNFαIL-22

IL-4IFNγ

IL-6

IL-4

IFNγ

IL-12 TGFβIL-6

IL-4 TGFβ

Inflammation &Autoimmunity

HumoralImmunity

Suppression

Th1

T-bet RORγt

GATA-3 Foxp3

Th17

IL-4IL-5

IL-13IL-25/IL-17E

TGFβIL-10

Th2 Treg

Thp

8

table 1. cytokine expression profile of human PBMcs after treatment with various stimulating agents. Human pBMCs were treated with LpS, Con A or pHA at 37 °C for 48 hr and cell-free samples were collected and assayed as described. Approximate cytokine responses are indicated as (+) 40 to 100 pg/mL, (++) 101 to 1000 pg/mL or (+++) >1000 pg/mL. Unstimulated samples were 0-40 pg/mL for most analytes.

table 2. cytokine response in human normal and diseased plasma samples. Human disease plasma samples were obtained from Bioreclamation and assayed in the Human Th17 panel as described. Values are the sample range in pg/mL for the indicated number of samples. Highlighted cytokines are Th17 cell-specific plasma cytokines.

Materials and methodsSample preparationPeripheral blood mononuclear cells (PBMCs,

Bioreclamation) were used as a representative immune

cell population that includes T helper cells. PBMCs were

thawed, washed and resuspended in RPMI Media (Gibco)

containing 10% fetal calf serum and 1% Penicillin/

Streptomycin at 106 cells per mL. The PBMCs were aliquoted

at 106 cells per well and incubated overnight at 37 °C. The

next day, either LPS, Con A or PHA (Sigma) were added for

final concentrations of 10, 20 or 20 µg/mL, respectively.

Human disease plasma and in vivo LPS-challenged mouse

plasma samples were obtained from Bioreclamation.

Immunoassay protocolThe multiplex assay was performed in a 96-well plate. The

plate was wet with 150 µL assay buffer for 10 min and

decanted. 25 µL standards or samples, 25 µL beads and

25 µL assay buffer or 25 µL matrix was added to the wells

and incubated overnight at 4 °C. Beads were washed twice,

then mixed with 25 µL biotinylated detection antibody

cocktail and incubated at room temperature (RT) for 1h.

25 µL Streptavidin-Phycoerythrin was added and further

incubated at RT for 30 min. Lastly, beads were washed

twice, mixed with 100 µL sheath fluid and analyzed on a

Luminex 200™ system.

resultsOur results demonstrated that LPS, Con A or PHA

treatments induced significantly increased secretion of

many cytokines, including Th17 cell-specific cytokines,

from human PBMCs (e.g. IL-6, TNFα, IL-17A, IL-17F and

IL-22, Table 1).

We examined cytokine levels in plasma samples from

humans with sepsis, RA, and lupus. These samples

displayed increased levels of Th17 cell-specific plasma

cytokines (Table 2).

cytokine response LPS con A PHA

IL-1β +++ ++ ++

IL-2 + +++ +++

IL-4 ++ +

IL-5 ++ +

IL-6 +++ +++ +++

IL-9 ++ ++

IL-10 ++ ++ ++

IL-13 +++ ++

IL-17A ++ ++

IL-17F +++ ++

IL-21 + +

IL-23 + +

IL-27 ++ + ++

IL-31 ++ ++

IFNg +++ +++

GM-CSF ++ +++ +++

MIP-3α ++ ++ ++

TNFα +++ +++ +++

TNFβ ++ ++

cytokine response Healthy (n=16) Sepsis (n=16) rA (n=4) Lupus (n=4)

IL-1β 0-7 0-129 0-143 0-30

IL-2 ND 0-160 0-514 0-108

IL-4 ND 0-735 0-3 ND

IL-5 0-12 0-88 0-5 ND

IL-6 4-16 31-703 0-373 0-11

IL-9 ND 0-185 0-143 ND

IL-10 ND 0-1603 0-2 0-8

IL-12p70 ND 0-126 0-185 0-23

IL-13 9-42 12-300 0-11 0-1

IL-15 ND 9-80 0-89 0-13

IL-17A ND 0-111 0-1 ND

IL-17E/IL-25 ND 0-12479 0-18634 0-951

IL-17F 30-230 15-1038 0-1237 0-79

IL-21 9-25 22-254 0-34 0-3

IL-22 ND 37-2042 0-729 ND

IL-23 ND 0-13183 0-12472 0-1685

IL-27 80-1000 2333-17179 505-8777 429-804

IL-28A 100-500 200-2974 0-1123 1-313

IL-31 0-65 0-4614 0-3013 0-17

IL-33 10-130 0-1202 0-320 0-10

IFNg ND 0-445 0-30 ND

GM-CSF 100-850 61-3262 0-1493 0-320

MIP-3α 6-55 48-1207 0-127 7-48

TNFα 5-12 20-4355 4-72 2-16

TNFβ ND 0-386 0-474 0-13

9

table 3. cytokine response of stimulated mouse PBMcs. Mouse pBMCs were treated with LpS, Con A or pHA at 37 °C for 48 hr, then cell-free samples were collected and assayed as described. Approximate cytokine responses are indicated as (+) 1 to 20 pg/mL, (++) 21 to 500 pg/mL or (+++) >500 pg/mL. Unstimulated samples were not detectable for most samples except IL-6 at approximately 30 pg/mL.

table 4. cytokine levels in the plasma of mice challenged with LPS for varying lengths of time. 8–12 week old cd-1 mice were injected IP with 1 mg/kg E. coli 055:B5 LPS (Sigma-Aldrich) suspension in saline. Plasma (disodium EdtA) was collected at various time points and assayed. data are pg/mL (SEM indicated in parentheses). n=8

Figure 2. Plasma cytokine levels in mice injected with LPS. 8-12 week old cd-1 Mice were injected IP with 1 mg/kg E. coli 055:B5 LPS (Sigma-Aldrich) suspension in saline, IP. Plasma (disodium EdtA) was collected at various time points and assayed for IL-17A, IL17F, IL-13 (A) and IL-6, IL-22, tNFα, IL-10 and IFNg (B). Error bars reflect standard error of the mean (N=8).

Using the MILLIPLEX® map Mouse Th17 Cytokine/Chemokine

Panel, we assessed the cytokine response in mouse PBMCs

after treatment with LPS, Con A and PHA. As in the case

of human PBMCs, the mouse PBMCs displayed a different

pattern of elevated cytokine secretion in response to LPS

compared to the pattern exhibited upon treatment with

Con A or PHA.

Multiplexed assays are extremely useful for assessing levels

of analytes in limited in vivo mouse samples, especially

given that only small amounts of blood can be drawn from

a mouse at one time. In this study, mice treated with LPS

for various time periods were assessed for in vivo cytokine

response with respect to treatment time. While IL-13 and

IL-6 were abundant in samples from mice treated up to 2 h,

these levels began to diminish with longer treatments. On

the other hand, levels of IL-17F began to increase with LPS

treatments of 4 h or more (Table 4 and Figure 2).

cytokine response LPS con A PHA

IL-1β +

IL-2 + +

IL-4 +

IL-5 +

IL-6 +++ ++ ++

IL-10 +

IL-17A ++ +

IL-17F ++ +

IL-22 +

MIP-3α +

TNFβ ++

Length of LPS treatment IL-1β IL-6 IL-10 IL-12p70 IL-13 IL-15

0 3 (3) 31 (22) 53 (16) 44 (17) 198 (55) 57 (5)

1 11 (3) 7648 (352) 3053 (591) 106 (38) 363 (58) 75 (17)

2 102 (46) 7129 (871) 1855 (516) 80 (12) 339 (30) 48 (5)

4 60 (13) 5542 (1017) 1192 (429) 77 (10) 108 (20) 44 (6)

8 20 (6) 2616 (921) 442 (93) 114 (29) 0 (0) 124 (48)

Length of LPS treatment IL-17A IL-17E IL-17F IL-22 IL-23 IL-27

0 45 (26) 3914 (1456) 40 (8) 158 (34) 460 (120) 1383 (494)

1 94 (14) 6049 (694) 15 (6) 190 (96) 962 (190) 2093 (535)

2 102 (11) 6392 (476) 82 (37) 923 (163) 680 (77) 2183 (433)

4 113 (21) 5044 (617) 379 (69) 1278 (250) 612 (70) 1961 (384)

8 132 (22) 3590 (510) 365 (151) 661 (84) 538 (48) 2805 (992)

Length of LPS treatment IL-28B cd40L GM-cSF IFNg MIP-3α tNFα

0 137 (15) 157 (24) 45 (20) 20 (12) 96 (19) 20 (6)

1 171 (38) 167 (48) 70 (12) 27 (10) 157 (73) 1871 (489)

2 239 (59) 131 (8) 188 (52) 38 (9) 3307 (758) 1017 (376)

4 199 (17) 110 (5) 57 (13) 1052 (370) 8360 (5979) 164 (42)

8 228 (45) 201 (26) 39 (12) 592 (246) 352 (61) 70 (20)

600

500

400

300

200

100

00 1 2

Time after LPS (hr)

Plas

ma

Leve

ls (p

g/m

L)

4 8

Mouse Plasma Cytokines

IL-13 IL-17F IL-17A

900080007000600050004000300020001000

00 1 2

Time after LPS (hr)

Plas

ma

Leve

ls (p

g/m

L)

4 8

Mouse Plasma Cytokines

IL-6 IL-22 TNF IL-10 IFNγ

A. B.

10

discussion and conclusionsUsing multiplexed assay panels, we were able to measure

large numbers of cytokine and chemokine analytes in

multiple samples simultaneously and obtain results that

were consistent with those previously reported. This

multianalyte profiling approach has the potential to generate

assays that can stratify groups of biological samples with

increased sensitivity and specificity compared to single-

plex assays, which quantify a single biomarker at a time.

Especially given that immune responses are very complex,

not only in normal, human systems but also in preclinical

models and in patients with chronic inflammation and

autoimmunity, these MILLIPLEX® map Th17 panels may

significantly accelerate cytokine profiling in particular and

immunology research in general.

references1. Murdaca, G et al. The role of Th17 lymphocytes in the

autoimmune and chronic inflammatory diseases. Intern Emerg Med. 2011 Dec;6(6):487-95.

2. Korn T, et al, IL-17 and Th17 Cells, Annu Rev Immunol 2009; 27:485-517.

3. Kolls, JK and Khader, SA. The role of Th17 cytokines in primary mucosal immunity. Cytokine & Growth Factor Reviews 2010; 21:443–448.

4. Blaschitz C and Raffatellu M. Th17 cytokines and the gut mucosal barrier. J Clin Immunol 2010; 30:196–203.

5. Wilke CM. et al. Deciphering the role of Th17 cells in human disease. Trends Immunol. 2011 Dec;32(12):603-11.

description catalogue No.

MILLIPLEX® map Human TH17 Magnetic Bead Panel HTH17MAG-14K

MILLIPLEX® map Mouse TH17 Magnetic Bead Panel MTH17MAG-47K

MILLIPLEX® map Human Cytokine/Chemokine Magnetic Bead Panel I HCYTOMAG-60K

MILLIPLEX® map Human Cytokine/Chemokine Magnetic Bead Panel II HCYP2MAG-62K

MILLIPLEX® map Human Cytokine/Chemokine Magnetic Bead Panel III HCYP3MAG-63K

MILLIPLEX® map Human High Sensitivity Cytokine/Chemokine Magnetic Bead Panel HSCYTMAG-60SK

MILLIPLEX® map Mouse Cytokine/Chemokine Magnetic Bead Panel MCYTOMAG-70K

MILLIPLEX® map Mouse Cytokine/Chemokine Magnetic Bead Panel II MCYP2MAG-73K

MILLIPLEX® map Mouse Cytokine/Chemokine Magnetic Bead Panel III MCYP3MAG-74K

Available from www.millipore.com.

RELATED pRoDUCTS

11

LentiBrite™ Lentiviral Biosensors for fluorescent cellular imaging: analysis of the cytoskeleton

Karyn Huryn-Selvar, Haizhen Liu, Janet Anderl, Jun Ma, and Luke ArmstrongEMD Millipore Corporation

AbstractReal-time analysis of cytoskeletal dynamics within live cells has been greatly enabled by expression of genetically encoded, fluorescently tagged proteins. In this study, we used LentiBrite™ lentiviral biosensors, which are high-titer, ready-to-use, pre-packaged lentiviral particles encoding cytoskeletal marker proteins tagged with GFP or RFP, for fluorescent imaging of cytoskeletal structures. Specifically, we used LentiBrite™ biosensors to visualize microtubules (α-tubulin), actin microfilaments (β-actin), microfilament cross-link sites (α-actinin), intermediate filaments (vimentin), and focal adhesions (paxillin). We demonstrated that these biosensors, which are validated for use with fixed and live-cell fluorescent microscopy, did not perturb the structures of interest. Localization of the GFP- or RFP-tagged proteins coincided with the endogenous proteins as determined by immunocytochemistry, and the biosensors displayed characteristic rearrangements upon treatment with known modulators of cytoskeletal structure. Thus, this panel of lentiviral biosensors provides a convenient method for visualization of cytoskeletal structure and dynamics under a variety of physiological and pathological treatment conditions, in both endpoint and real-time imaging modalities.

IntroductionAnalysis of the dynamics of subcellular structures has been revolutionized in the past 15 years by the development and refinement of genetically-encoded fusions between fluorescent proteins and cellular structural proteins. Such fusion proteins, if designed properly, incorporate into the structure of interest without disturbing its function, and permit visualization of the structure in live cells in real time by fluorescence microscopy1. Traditionally, cDNAs encoding the fusion proteins are delivered into cells by chemical transfection or electroporation. However, such transient transfection procedures have drawbacks, including highly variable expression levels and low efficiencies for transfecting primary cells. Selection of clonal cell lines stably expressing the construct of interest allows for optimized expression levels, but the process is time-consuming and is not feasible for primary cells. Fortunately, recently developed viral gene delivery

vectors, such as lentiviral and adenoviral vectors, permit transduction of virtually any cell type, at more tightly controlled expression levels. Although viral vectors have been successfully used to express genetically encoded subcellular markers, prepackaged viral vectors have not been widely available, and researchers have had to perform packaging procedures themselves in order to perform such experiments.

In a previous report, we introduced the LentiBrite™ product line of prepackaged lentiviral particles encoding fluorescent fusion proteins with subcellular markers for cell fate and cytoskeletal structures2. The lentivirus particles are packaged with 3rd generation technology, which produces pseudoviral particles that have vanishingly low probabilities of pathogenicity3. The fluorescent proteins employed are TagGFP2 and TagRFP, which have been demonstrated to be monomeric for minimal interference with the function of the fusion partner proteins, and have quantum yields comparable to fluorescent proteins from other species4,5. Furthermore, transduction of cells with these lentiviral vectors offers higher transfection efficiency and more homogeneous protein expression than by other non-viral transfection methods, particularly for traditionally difficult-to-transfect primary cell types.

In this article, we highlight the use of lentiviral biosensors for analyzing dynamics of the major cytoskeletal structures, including focal adhesions, actin microfilaments, microfilament crosslink sites, intermediate filaments, and microtubules. These biosensors contain TagGFP2 and TagRFP and are fused at their N- or C-termini to specific components of the cytoskeletal protein of interest. Focal adhesions are detected by fluorescent protein fusions to paxillin. Actin microfilaments are highlighted by fusions of the fluorescent proteins to the N-terminus of β-actin. Sites of actin microfilament cross-bridging and microfilament-focal adhesion interaction are revealed with fluorescent protein fusions to α-actinin. Microtubules are detected by fusions of FPs to the N-terminus of α-tubulin, such that structurally important modifications to the tubulin C-terminus are unhindered. Finally, intermediate

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10 to 40. Infected cells were then incubated at 37 °C, 5% CO2 for 24 h. 24 h after lentiviral transduction, lentivirus-containing medium was removed and replaced with fresh growth medium. All lentivirus-containing media and plasticware in direct contact with virus-containing solutions were disinfected with 10% bleach before disposal. Cells were cultured for another 24-48 h, with media changed every 24 hours. For inhibitor experiments, cells were incubated in the inhibitor of interest at the concentration and for the time indicated in the legend.

Live cell imagingFor live cell visualization, the growth medium was replaced with Dulbecco’s Modified Eagle Mediumgfp (DMEMgfp, Evrogen) containing 10% fetal bovine serum (FBS) and 25 mM HEPES. The chambered cover glass was placed in a temperature-controlled microscope stage insert upon a Leica DMI6000B inverted wide-field fluorescent microscope with a 63X oil-immersion objective lens and illumination/filters appropriate for GFP or RFP visualization. Imaging was initiated as rapidly as possible following the addition of modulators.

cell fixation, staining and imagingFor end-point imaging, cells in chamber slides were fixed for 30 minutes at room temperature with 3.7% formaldehyde in Dulbecco’s phosphate-buffered saline (DPBS). During fixation and for all subsequent steps, cells were protected from light to minimize photobleaching. For immunocolocalization studies, samples were then rinsed twice with fluorescent staining buffer (DPBS with 2% blocking serum and 0.25% Triton® X-100). Primary antibody in fluorescent staining buffer was added to each well and incubated 1h at room temperature. Samples were then rinsed three times with fluorescent staining buffer before incubation for 1h at room temperature with fluorescent secondary antibody and DAPI (1 µg/mL) in staining buffer. Finally, samples were rinsed twice each with fluorescent staining buffer and DPBS, and slides were coverslipped with mounting medium containing anti-fade reagent and No. 0 cover glasses (Ted Pella). Mounted specimens were imaged on inverted wide-field (as above) or Leica DMI4000B confocal fluorescent microscopes, utilizing illumination and filters appropriate for GFP, RFP, Cy5 (for immunocolocalization), or DAPI excitation and emission wavelengths. Imaging was performed with a 63X oil-immersion objective lens unless otherwise indicated.

filaments are highlighted with fusions to vimentin. All of these cytoskeletal biosensor lentiviral particles provide bright fluorescence and precise localization to enable live cell analysis of cytoskeletal dynamics in difficult-to-transfect cell types.

Materials and methodsconstruction of lentiviral vectors encoding fluorescent protein fusionsThe cDNAs encoding TagGFP2 and TagRFP were obtained from Evrogen. LentiBrite™ RFP- and GFP-α-tubulin were constructed by cloning the full-length cDNA sequence of the human tubulin α-1B at the C-terminus of the fluorescent protein cDNA. LentiBrite™ RFP- and GFP-β-actin were constructed by cloning the full-length cDNA sequence of human β-actin at the C-terminus of the fluorescent protein cDNA. LentiBrite™ GFP- and RFP-vimentin were constructed by cloning the full-length cDNA sequence of the human vimentin at the C-terminus of the fluorescent protein cDNA. LentiBrite™ α-actinin-GFP and -RFP were constructed by cloning the full-length cDNA sequence of human α-actinin-1 isoform b at the N-terminus of the fluorescent protein cDNA. LentiBrite™ paxillin-GFP and -RFP were constructed by cloning the full-length cDNA sequence of human paxillin at the N-terminus of the fluorescent protein cDNA. Constructs were transferred to pCDH-EF1-MCS (System Biosciences), a lentiviral vector containing the constitutive, moderately expressing EF1α promoter. 3rd generation HIV-based VSV-G pseudotyped lentiviral particles were generated using the pPACKH1 Lentivector Packaging System at System Biosciences Inc. (www.systembio.com).

cell seeding and lentiviral transductionCells in growth medium were seeded onto 8-well glass chamber slides (Millicell® EZ slides) for fixed cell imaging, or chambered cover glasses for live cell imaging. Seeding densities were selected to provide for 50-70% confluency after overnight culture (e.g., 20,000-40,000 cells/cm2). The next day after seeding, medium was replaced with fresh growth medium. High-titer lentiviral stock was diluted 1:40 with growth medium, and appropriate volumes of lentivirus were added to the seeded cells to achieve the desired multiplicity of infection (MOI). MOI refers to the ratio of the number of infectious lentiviral particles to the number of cells being infected. Typical MOI values ranged from

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results and discussionImproved biosensor expression efficiency with lentiviral transductionWe constructed a panel of lentiviral particles containing genetically encoded fluorescent markers for cytoskeletal elements. By using these lentiviral biosensors, both transfection efficiency and homogeneity were greatly improved relative to chemical transfection with plasmid DNA (Figure 1). First, easily transfectable HeLa cells were transfected with GFP-labeled tubulin using either plasmid DNA or by lentivirus. Lentivirally-transduced cells demonstrated higher transfection efficiency (percentage of cells positive for signal, compared to total number of cells), as well as more homogeneous expression (compared to the range of high and low expressers in the chemically transfected population). For a typically “hard-to-transfect” primary cell type such as human umbilical vein endothelial cells (HUVEC), lentiviral transduction produced homogeneously bright signal in a significant proportion of cells, in contrast to chemical plasmid transfection, which resulted in minimal GFP-tubulin expression.

Panel of lentiviruses encoding biosensors for cytoskeletal elementsSpecificity of subcellular localization of each of the cytoskeleton-directed biosensors is presented in Figure 2. Transduction of REF-52 rat fibroblasts with the paxillin-GFP lentiviral particles resulted in selective localization at focal adhesions, which appear as short linear streaks present predominantly but not exclusively at the periphery of the cell (Figure 2A). In contrast, lentiviral transduction of REF-52 with α-actinin-RFP presented a pattern of parallel, striated microfilaments, consistent with the role of α-actinin in crosslinking individual actin microfilaments (Figure 2B). REF-52 cells lentivirally transduced with β-actin-RFP revealed a similar array of parallel microfilaments spanning the length of the cell, except these filaments were continuously fluorescent along their entire lengths (Figure 2C). HUVECs transduced with lentivirus encoding RFP-α-tubulin also displayed a filamentous pattern of fluorescence, except these filaments extended from a central point adjacent to the nucleus, consistent with the well-described microtubules extending from the microtubule-organizing center (Figure 2D). GFP-vimentin expressed in human mesenchymal stem cells (huMSC) by means of lentiviral transduction highlighted a pattern of filaments weaving from end to end of the spindle-shaped cell, as expected for intermediate filaments (Figure 2E).

dAPI GFP-tubulin dAPI GFP-tubulinGFP-tubulin GFP-tubulinHeLa (easy-to-transfect)

Chemical transfection

LentiBrite™ lentiviral

transduction

HuVEc (difficult-to-transfect)

Figure 1. Plasmid vs. lentivirus transfection in easy- and difficult-to-transfect cell types. HeLa cells and HUVECs were transfected with a TagGFp2-tubulin-encoding construct, either utilizing plasmid DNA in conjunction with a lipid-based chemical transfection reagent, or using LentiBrite™ lentiviral particles. Images were obtained via wide-field fluorescent imaging with a 20X objective lens (blue = DApI nuclear counterstain, green = GFp-tubulin). Lentiviral transduction resulted in higher transfection efficiency (particularly for HUVEC, for which plasmid transfection was unsuccessful) and GFp-tubulin signal of more uniform fluorescence intensity.

Figure 2. LentiBrite™ lentiviral biosensors localize to specific cytoskeletal elements. Cells were transduced with the following biosensors and imaged by wide-field fluorescence: (A) GFp-paxillin highlights focal adhesions in REF-52 cells. (B) α-actinin-RFp in REF-52 cells displays a striated filamentous pattern corresponding to actin microfilament cross-link sites. (C) β-actin-RFp in REF-52 cells displays a similar filamentous pattern to that of α-actinin, but with a pattern of continuous intensity. (D) RFp-α-tubulin in HUVECs displays a pattern of microtubule filaments radiating from the microtubule organizing center (white arrow). (E) GFp-vimentin in huMSCs highlights the intermediate filaments.

A.

d.

B.

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c.

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Specificity of localization of lentivirally delivered biosensorsAs genetically encoded biosensors can be prone to improper localization, due either to a propensity of the particular fluorescent protein to aggregate or to the overexpressed biosensor over-saturating the structure of interest, we sought to confirm the specificity of lentivirally delivered biosensor localization. We found that lentiviral delivery of fluorescent protein-tagged cytoskeletal markers enabled accurate detection of the appropriate cytoskeletal elements, as determined by 1) redistribution of the biosensor upon treatment of the cells with specific inhibitors of the cytoskeletal structure, 2) similar distribution of the biosensor to endogenous protein as determined by immunofluorescence, and 3) live cell imaging of cytoskeletal dynamics.

Microtubules present a useful cytoskeletal element for inhibitor-mediated modulation of structure, due to the availability of small molecules that either promote microtubule disassembly or cause aberrant stabilization of the microtubules. In Figure 3, HUVECs were lentivirally transduced with RFP-tubulin, and treated with the microtubule stabilizer, paclitaxel, or the microtubule depolymerizer, nocodazole. As previously shown, the untreated cells were characterized by a prominent perinuclear microtubule organizing center with radiating microtubules (Figure 3A). Upon treatment with paclitaxel, the microtubules formed large bundles that wrapped around the nucleus (Figure 3B). In contrast, treatment with nocodazole resulted in a complete loss of both microtubules and microtubule organizing center (Figure 3C).

The interconnected nature of intermediate filaments with microtubules is demonstrated in Figure 4. HeLa cells transduced with lentivirus encoding RFP-vimentin ordinarily display a meshed pattern that both encircles the nucleus and extends into the cytosol (Figure 4A). Upon treatment of the cells with nocodazole, the intermediate filaments collapsed into a perinuclear mass, in contrast to the dissipation of microtubule structure (Figure 4B). Detection of vimentin by immunocytochemistry confirmed that endogenous vimentin assumed the same filamentous pattern as the GFP-vimentin, and underwent the same rearrangement upon nocodazole treatment. The dependency of the meshwork arrangement of intermediate filaments upon microtubule integrity has previously been demonstrated by immunocytochemistry, and has been demonstrated to be mediated by association of intermediate filament proteins with the microtubule motor proteins, kinesins6.

A. B. c.

Figure 3. Localization of rFP-tagged tubulin following modulator treatment. HUVECs were lentivirally transduced with RFp-tubulin biosensors at an MoI of 40. Transduced cells were subjected to the following treatments for 4 h: (A) untreated, (B) treated with 1 µM paclitaxel, or (C) treated with 25 µM nocodazole. Cells were fixed, mounted, and imaged by wide-field fluorescence microscopy. Cells treated with paclitaxel displayed bundled microtubules that curved around the nucleus, whereas cells treated with nocodazole contained no evident microtubule structures.

Figure 4. Fluorescent protein expression and colocalization with fluorescent antibody staining. HeLa cells were transduced with TagRFp-vimentin biosensors at an MoI of 20, and 72 h later, either left untreated or treated with 25 µM nocodazole. Cells were subsequently fixed, immunostained with an anti-vimentin antibody, and imaged by wide-field microscopy. Untreated cells displayed a filamentous vimentin network, in contrast to a clumped perinuclear distribution in cells treated with nocodazole. In both cases, the fluorescently tagged protein (top and middle rows) colocalized with staining obtained with anti-vimentin antibody (bottom row).

untreated Nocodazole

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Figure 5. Live cell time-lapse imaging of lentivirally-transduced cells. REF-52 cells were lentivirally transduced with TagRFp-β-actin biosensors, and imaged by oil-immersion wide-field microscopy in real time. The cells were dosed with 2 µM cytochalasin D, and imaging was immediately initiated, with images obtained every 30 s for 30 min. Still-frame captures demonstrated transition of fine filamentous organization to a granular pattern in the vicinity of the nucleus, and diffuse distribution at the cellular periphery. Full-length videos of live cell imaging using LentiBrite™ Lentiviral Biosensors are available at www.millipore.com/cellstructure.

We also characterized the effect of the actin microfilament destabilizer, cytochalasin D, on lentivirally expressed RFP-β-actin in REF-52 cells. Still-frame captures of modulator-treated cells at various time-points are provided in Figure 5. By wide-field microscopy, cells were imaged every 30 seconds over the course of 10 minutes following addition of the inhibitor. Within 5 minutes of treatment, the microfilaments broke into fine aggregates in the center of the cell, or dissipated into a diffuse distribution in the cellular periphery. This pattern of disruption of actin microfilaments by cytochalasins is consistent with prior observations and results from the ability of cytochalasins to cap and sever actin microfilaments and to prevent assembly of G-actin into microfilaments7.

conclusionLentiviral biosensors enable convenient transduction of easy- and difficult-to-transfect cell types with fluorescent protein-tagged subcellular markers. These LentiBrite™ pre-packaged lentiviral particles enable higher efficiency and more homogeneous expression of introduced proteins compared to nonviral transfection methods. We have demonstrated the use of GFP- or RFP-tagged cytoskeletal markers in both fixed and live cell microscopy applications. The encoded proteins displayed colocalization with antibody staining and appropriate redistribution upon treatment with known modulators of cytoskeletal structure. LentiBrite™ biosensors provide a ready-to-use solution for researchers seeking to fluorescently visualize the presence, absence or trafficking of a protein, under normal, abnormal, diseased, or induced cellular states.

references1. Goldman, R.D., Swedlow, J.R., and Spector D.L. (Editors). Live

Cell Imaging: A Laboratory Manual, 2nd ed., 2009; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

2. Anderl, J. et al. LentiBrite™ lentiviral biosensors for fluorescent cellular imaging: analysis of autophagosome formation. EMD Millipore Cellutions. 2011; Vol. 4:25-30.

3. Pauwels, K. et al. State-of-the-art lentiviral vectors for research use: risk assessment and biosafety recommendations. Curr. Gene Ther. 2009; 9: 459-474.

4. Mertzlyak, E.M. et al. Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat. Methods 2007; 4: 555-557

5. Subach, O.M. et al. Conversion of red fluorescent protein into a bright blue probe. Chem. Biol. 2008; 15: 1116-1124.

6. Chang, L. and R.D. Goldman. Intermediate filaments mediate cytoskeletal crosstalk. Nat. Rev. Mol. Cell Biol. 2004; 5: 601-613.

7. Spector, I et al. New anti-actin drugs in the study of the organization and function of the actin cytoskeleton. Microsc. Res. Tech. 1999; 47: 18-37.

description catalogue No.

LentiBrite™ Paxillin-GFP Lentiviral Biosensor 17-10154

LentiBrite™ Paxillin-RFP Lentiviral Biosensor 17-10155

LentiBrite™ α-Actinin-GFP Lentiviral Biosensor 17-10156

LentiBrite™ α-Actinin-RFP Lentiviral Biosensor 17-10196

LentiBrite™ GFP-Tubulin Lentiviral Biosensor 17-10206

LentiBrite™ RFP-Tubulin Lentiviral Biosensor 17-10205

LentiBrite™ GFP-β-Actin Lentiviral Biosensor 17-10204

LentiBrite™ RFP-β-Actin Lentiviral Biosensor 17-10203

LentiBrite™ GFP-Vimentin Lentiviral Biosensor 17-10152

LentiBrite™ RFP-Vimentin Lentiviral Biosensor 17-10153

LentiBrite™ GFP-LC3 Lentiviral Biosensor 17-10193

LentiBrite™ RFP-LC3 Lentiviral Biosensor 17-10143

LentiBrite™ GFP-LC3 Control Mutant Lentiviral Biosensor

17-10189

LentiBrite™ RFP-LC3 Control Mutant Lentiviral Biosensor

17-10188

LentiBrite™ Calreticulin-RFP-KDEL Lentiviral Biosensor

17-10146

LentiBrite™ GFP-HMGB Lentiviral Biosensor 17-10147

Millicell® EZ SLIDE (4-well) PEZGS0416

Millicell® EZ SLIDE (8-well) PEZGS0816

DAPI, Dihydrochloride 268298

EndoGRO® Human Umbilical Vein Endothelial Cells (HUVEC)

SCCE001

Human Mesenchymal Stem Cell Kit (Derived from Bone-Marrow)

SCR108

Polybrene Infection/Transfection Reagent TR-1003-G

Nocodazole 487928

Paclitaxel 580555

Cytochalasin-D 250255

Jasplakinolide 420107

Actin Polymerization Interfering Agents Set 104850

Mouse Anti-Vimentin, clone V9 MAB3400

Available from www.millipore.com.

RELATED pRoDUCTS

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IntroductionExpression of eukaryotic genes during development requires

complex spatiotemporal regulation, which is often achieved

through the coordinated interaction of regulatory elements

with its enhancers and promoters. The identification and

mapping of regulatory elements on a genome-wide scale is

crucial for understanding how gene expression is regulated.

Chromatin immunoprecipitation is a standard method for

assessing the DNA-protein interactions in vivo, in a native

chromatin context. However, conventional chromatin

immunoprecipitation protocols are time-consuming, labor-

intensive, and unsuitable for analyzing multiple samples

simultaneously.

Recently, we developed a simple, high throughput chromatin

immunoprecipitation protocol that uses high-capacity,

protein A/G-coated magnetic beads and 96-well plates. This

protocol has fewer steps, requires less hands-on time, and

has higher sensitivity and more reliable performance than

conventional approaches. The method is also compatible

with multichannel pipetting as well as liquid handling

systems. We have successfully used this protocol to map

various clinically relevant chromatin marks and controls

across several cell types, to quantitatively measure

chromatin states. This analysis included a variety of marks

corresponding to promoters, enhancers, putative insulators

and transcribed regions, as well as large-scale repressed

and inactive domains. Here we demonstrate how the HT96

protocol can be used to characterize model cellular systems.

MethodsThe detailed protocol for these experiments can be found in

the Magna ChIP™ HT96 Kit manual (Catalogue No.

17-10077). Briefly, cells and tissues were crosslinked with

1% formaldehyde, and nuclei were isolated and sonicated

to produce chromatin fragments of approximately 200 to

600 bp. The antibodies used were incubated with magnetic

protein A/G beads in a 96-well plate at 4 °C for 2 h, and ChIP

reactions were performed overnight at 4 °C, followed by

washing with high and low stringency buffers. Quantitative

PCR (qPCR) analysis with SYBR® green fluorophore was

performed with ChIP DNA following protein digestion and

crosslink reversal. qPCR data analysis was performed using

the relative standard curve method.

Cellular characterization of chromatin state via high throughput ChIp assay Zirong Li, tracy cooke, Hugh Spotswood, Konstantin taganov, Bhaskar thyagarajan, John rosenfeld ,and robert Kovelman EMD Millipore Corporation

Cells Tissues

In vivo cross-linkingLysis

Isolation of chromatincultured cells or tissues

Sonication to shearchromatin

96-well plate immunoprecipitation• 10,000-100,000 cells per reaction• Manual or automated processing

Reversal of cross-links

DNA purification (optional)

Detection• Quantitative PCR• Promoter microarray• Sequencing

Amplification2000

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RFU

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Figure 1. Illustrated workflow for plate-based, high-throughput chIP assay

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resultsPerformance with tissue samples and low cell numbers Independent ChIP analyses show that the Magna ChIP™

HT96 protocol produces exceptional signal-to-noise ratio

with as few as 10, 000 cells (Figure 2), and performs better

than a leading competitor’s kit (Figure 3). This kit also

performs well with tissues (Figure 4).

Next, we used the HT90 kit to analyze the enrichment of

seven proteins or modification states on different DNA-

binding regions in various cell and tissue samples (Figure

5). An experiment of this scale would have been quite time-

consuming using traditional ChIP protocols; in contrast,

the the Magna ChIP™ HT96 kit enabled high throughput

sample analysis and facilitated side-by-side comparison of

chromatin-binding patterns across samples.

Figure 2. Effective enrichment of rNA Pol II on GAPdH promoter using only 10,000 cells. Sonicated chromatin prepared from 10,000 HeLa cells was subjected to chromatin immunoprecipitation using 1 µg of purified mouse IgG (Catalogue No. 12-371B), and 1 ug of anti-RNA pol II (Catalogue No. 17-620), and the Magna ChIp™ HT96 kit. Immunoprecipitation of antibody-associated DNA fragments was verified by qpCR using control primers flanking the human GApDH promoter region. Error bars represent standard deviation of qpCR triplicates.

Figure 3. Superior enrichment compared to a high throughput chIP kit from another supplier. Sonicated chromatin prepared from 10,000 HeLa cells was subjected to chromatin immunoprecipitation using 1 µg of purified rabbit IgG (Catalogue No. 12-370) or 0.1 µg of anti-H3K4Me3 (Catalogue No. 17-614) and the Magna ChIp™ HT96 kit or Competitor D kit. Relative enrichment of H3K4me3 at human GApDH promoter region was measured by qpCR. Error bars represent standard deviation of qpCR triplicates.

Figure 4. Enrichment of rNA Pol II on GAPdH promoter from 1 mm3 mouse brain tissue. Sonicated chromatin prepared from 1 mm3 of mouse brain tissue was subjected to chromatin immunoprecipitation using 1 µg of purified mouse IgG (Catalogue No. 12-371B), 1 µg of anti-RNA pol II (Catalogue No. 17-620), and the Magna ChIp™ HT96 Kit. Immunoprecipitation of antibody-associated DNA fragments was verified by qpCR using control primers flanking the human GApDH promoter region. Error bars represent standard deviation of qpCR triplicates.

Figure 5. Analysis of multiple chromatin samples in parallel using multiple antibodies. Chromatin was derived from sources indicated and subjected to immunoprecipitation with either specific ChIpAb+™ antibodies or control IgG (x-axis), using the Magna ChIp™ HT96 multichannel pipette protocol. Assays were performed using conditions and genomic locations described in the respective ChIpAb+™ product user guides.

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Performance with automated platformsTo assess the assay’s effectiveness and precision when it is

conducted using automated liquid handling platforms, we

quantified enrichment of RNA Poll II, phosoho-CREB, and

H3K4Me3 occupancy of the GAPDH promoter. As shown

in Figure 6A, conducting the Magna ChIP™ HT96 assay

on Tecan Freedom EVO® robotic workstation resulted in

significant enrichment of RNA Pol II and phospho-CREB at

GAPDH promoter. Figures 6B and 6C show good intraplate

and interplate reproducibility using this platform in

combination with the HT96 kit.

Efficient performance in cell-based assaysUsing antibodies to various histone modifications along with

the Magna ChIP™ HT96 kit, we assessed the modification

status of histones in various cellular contexts. First, we

found that acetylation of histone 3, lysine 9 at the P21

promoter increased dramatically upon exposure of U2OS

cells to ultraviolet (UV) light (Figure 7). Under the same

conditions, methylation of histone 3 lysine 4 increased

slightly and methylation of histone 3 lysine 27 (a repressive

histone mark) remained undetectable. P21 regulates the DNA

damage response, so it was not surprising that exposure to

UV light would result in an increase in histone modifications

associated with transcriptional activity.

Figure 6. High inter- and intra-assay reproducibility when using automated liquid handlers, such as the tecan workstation, with as few as 10, 000 cell equivalents. Sonicated chromatin prepared from 10,000 HeLa cells was subjected to chromatin immunoprecipitation using 1 µg of purified mouse IgG (Catalogue No. 12-371B) or rabbit IgG (Catalogue No. 12-370) and specific antibodies (RNA pol II, Catalogue No. 17-620; phospho-CREB (Catalogue No. 17-10131), Figure 5A; H3K4Me3, Catalogue No.17-614, Figures 5B and 5C) and the Magna ChIp™ HT96 Kit. Immunoprecipitations were conducted using a Freedom EVo® robotic workstation. Immunoprecipitation of antibody-associated DNA fragments was verified by qpCR using control primers flanking the human GApDH promoter region. Error bars in represent standard deviation of ChIp triplicates in the same plate (A and B) (technical replicates), or ChIp triplicates of each sample from three different plates (C) (biological replicates).

Figure 7. uV treatment (50 µjoules/15 cm plate) affects histone modification status at the P21 promoter in u2oS cells. Chromatin was derived from either untreated or UV treated U2oS cells. Immunoprecipitations were performed using the multichannel protocol of the Magna ChIp™ HT96 kit with various antibodies recognizing histone modifications or IgG as indicated. Relative enrichment of various histone modifications at the p21 promoter was measured by qpCR.

1.2

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H3K4me3 H3K27me3H3K9Ac

U2OSUV Treated U2OS

Rabbit IgG

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2.00.3 0.7 0.7

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0.0 0.0 0.0 0.00.0

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Figure 8. Effects of the pan HdAc inhibitor sodium butyrate (5 mM for 24 hours) on silent genes in HeLa cells. Chromatin was derived from either untreated or sodium butyrate treated HeLa cells. Immunoprecipitations were performed using the multichannel protocol of Magna ChIp™ HT96 kit with H3K9Ac antibody. Relative enrichment of H3K9Ac at various promoters (β-GLoBIN, HoXA7, and MYoD) was measured by qpCR.

Figure 9. Profiling of multiple chromatin states associated with H3K4me, H3K4me3, H3K9Ac, H3K27me3, H3K36me3, and ctcF. Chromatin was derived from HeLa cells. Immunoprecipitations were performed using the multichannel protocol of the Magna ChIp™ HT96 kit with various antibodies (H3K4me, H3K4me3, H3K9Ac, H3K27me3, H3K36me3 and CTCF). Relative enrichment of histone modifications and CTCF biding at various chromatin regions (promoter, coding region, enhancer, insulator and heterochromatin) were measured by qpCR. Data were normalized and displayed according to relative enrichment.

Next, we confirmed that treating cells with a histone

deacetylase (HDAC) inhibitor increased the enrichment

of acetylated histones at promoters of inactive genes

(normally deacetylated). After treating HeLa cells with

sodium butyrate, a HDAC inhibitor, we analyzed histone

acetylation at β-globin, HOXA7, and MYOD promoters. In all

cases, histone acetylation increased relative to the untreated

condition (Figure 8).

In addition to facilitating the analysis of chemical

perturbation on multiple chromatin sites, plate-based ChIP

also enabled the profiling of multiple types of chromatin

regions (such as promoters, enhancers, and coding regions),

in terms of enrichment of various associated proteins or

histone modification states (Figure 9). As expected, we found

that promoters in non-senescent HeLa cells were more

enriched in the permissive trimethylated histone 3 lysine

4 (H3K4me3) than in unmethylated histone 3 lysine 4 or

trimethylated histone 3 lysine 27, which are both associated

with repressed transcription. Notably, the transcriptional

repressor and zinc finger protein CTCF was very highly

enriched at insulator regions of chromatin, as previously

reported1. Insulator regions function to block the interactions

between enhancers and promoters. Almost as dramatic was

the enrichment of acetylated histone 3 lysine 9 at enhancer

regions, consistent with published epigenomic studies of

acetylation2.

2.5

Perc

ent o

f Inp

ut

1.5

2.0

β-GLOBIN MYODHOXA7

UntreatedTreated

1.0

0.5

0.0

0.4

1.4

0.50.1

2.4

0.3

H3K4me

H3K4me3

H3K9Ac

H3K27me3

H3K36me3

CTCF High

Low

Prom

oter

Codi

ng

Enh

ance

r

Inso

lato

r

Het

er C

hrom

atin

20

conclusionWe have developed a new approach that transforms ChIP from a labor-intensive, single-tube

process into a high throughput, plate-based process that is fully compatible with automated

liquid handling systems. The Magna ChIP™ HT96 kit provides a complete set of optimized

reagents for up to 96 ChIP reactions, and works well with as few as 10,000 cells or 1 mm3

of tissue. In contrast to a standard ChIP experiment that typically uses four buffers, this

protocol uses a single buffer for sonication, immunoprecipitation, and wash. With these

improvements to traditional ChIP, the Magna ChIP™ HT96 protocol greatly facilitates the

analysis of multiple chromatin samples and antibodies in a single experiment.

Given that effects of epigenetic marks on systems-level outcomes may be combinatorial

in nature, it is important to simultaneously analyze multiple epigenetic parameters in each

experimental condition. This high throughput ChIP protocol’s compatibility with minimal

sample amounts can increase the sensitivity and spatiotemporal resolution of the ChIP assay.

Thus, not only can this protocol accelerate chromatin analysis, but it can also enable deeper

insights into gene regulation.

references1. Cuddapah S et al. Global analysis of the insulator binding protein CTCF in chromatin barrier

regions reveals demarcation of active and repressive domains. Genome Res. 2009; 19 (1): 24–32.2. Roh TY et al. Genome-wide prediction of conserved and nonconserved enhancers by histone

acetylation patterns. Genome Res. 2007 Jan;17(1):74-81.

description catalogue No.

Magna ChIP™ HT96 Chromatin Immunoprecipitation Kit 17-10077

EZ-Magna ChIP™ HT96 Chromatin Immunoprecipitation Kit 17-10078

Magna GrIP™ HT96 Rack (for 96-well plates) 17-10071

description catalogue No.

ChIPAb+™ RNA Pol II 17-620

Normal Rabbit IgG 12-370

ChIPAb+™ Trimethyl Histone 3 Lysine 4 17-614

ChIPAb+™ phospho-CREB 17-10131

ChIPAb+™ E2F3 17-10062

ChIPAb+™ LSF 17-10252

ChIPAb+™ H3K9Ac 17-658

ChIPAb+™ FoxA2 17-10258

ChIPAb+™ H3T11P 17-10139

ChIPAb+™ JMJD1C 17-10262

Available from www.millipore.com.

Available from www.millipore.com.

oRDERING INFoRMATIoN

RELATED pRoDUCTS

21

IntroductionMammalian Hedgehog proteins include Sonic Hedgehog

(Shh), Indian Hedgehog (Ihh), and Desert Hedgehog (Dhh).

They are involved in regulating pattern formation during

embryonic development and in the maintenance of some

tissue types in adults. Shh is expressed mainly in epithelia

in teeth, hair, whiskers, gut, bladder, urethra, vas deferens,

and lung. Dhh is found in Schwann and Sertoli cell

precursors, and Ihh is expressed in gut and cartilage.

Molecular structureShh is the best-characterized Hedgehog protein. It is

synthesized as a 45 kDa precursor protein, which is then

autocatalytically cleaved to generate a 20 kDa N-terminal

fragment that is responsible for all Hh biological activity

and a 25 kDa C-terminal fragment that contains the

autoprocessing unit. The N-terminal fragment of Shh

contains palmitic acid and cholesterol as two lipid tethers,

which allow it to remain associated with the plasma

membrane. The cholesterol moiety is believed to be

responsible for directing Hedgehog traffic in the

secretory cells.

developmental rolesShh, a secreted morphogen, has been implicated in

several embryonic developmental processes. Shh

signaling is required throughout embryonic development

and is involved in the determination of cell fate

and embryonic patterning during early vertebrate

development. Disruption of Shh in humans also leads to

holoprosencephaly (lack of development of forebrain in

the embryo). Shh is involved in neural tube patterning

and in the development of left-right symmetry. In skin,

Shh is involved in maintaining the stem cell population.

Shh displays inductive, proliferative, neurotrophic, and

neuroprotective properties. Shh often works in concert

with the Wnt signaling protein in setting embryonic

patterns. During the late stage of development, Shh

is involved in the proper formation of a variety of

tissues and organs and it functions with other signaling

molecules, such as the fibroblast growth factors and

bone morphogenetic protein, to mediate developmental

processes.

Proteins involved in Shh signalingShh often works in concert with the Wnt signaling protein

in setting embryonic patterns. The Wnt pathway uses

β-catenin to transduce its signals to the nucleus; however,

the Shh pathway utilizes a 155 amino acid protein, Cubitus

interruptus (Ci155) in Drosophila or Gli in mammals. Shh

signaling is known to occur through a receptor complex

associating two membrane proteins, Patched (Ptc) and

Smoothened (Smo). Ptc is a twelve-pass membrane protein

that acts as a receptor and binds Hedgehog ligand; Smo

is a seven-pass membrane protein that acts as a signal

transducer. In this regard, Smo displays homology to

G-protein-coupled receptors that are usually associated

with G-protein-coupled receptor kinases.

Sonic Hedgehog: its dual role in morphogenic and mitogenic signalingchandra MohanEMD Millipore Corporation

Nucleus

GSK-3β

Shh

PKA

Gli

CKI

sufu

GliDyrk1

Gli responsive gene transcription

CREB

Ci155/Gli

Ub

Ub Ub

Ub

Proteasome

PTC SMO PTC

22

Signaling events in the Shh pathwayIn the absence of Shh, Ptc interacts with Smo and inhibits

its activity. Under these conditions, Ci is targeted for

proteolysis, which generates a truncated 75-amino acid

form (Ci75), which acts as a transcriptional repressor.

In vertebrates, three Gli proteins (Gli1, Gli2, and Gli3)

have been reported. Despite several homologous regions,

including a DNA-binding domain with five C2H2 zinc

fingers and a C-terminal transcription activation domain,

these three proteins have distinct activities and are not

considered to be functionally equivalent. Gli1 acts as a

transcriptional activator and Gli3 as a repressor. Gli2 can

act either as an activator or a repressor depending upon

post-transcriptional and post-translational modifications.

Shh binding to Ptc removes the inhibitory effect on

Smo and allows Ci/Gli to enter the nucleus and act as a

transcriptional activator. Smo action is mediated through

a protein complex containing the kinesin-like protein

Costal2 (Cos2), the Ser/Thr kinase Fused (Fu) and Ci/Gli.

Transcriptional activity of Ci/Gli is also regulated through

its binding to Suppressor of Fu (Sufu), which is a negative

regulator of Hedgehog signaling in Drosophila as well as in

vertebrates. It binds to all three Gli proteins with different

affinities. Shh controls cell cycle progression by regulating

the expression and activity of cyclins. It is also involved in

expression of EGF and EGF receptor.

Phosphorylation events in Shh signalingProtein kinase A (PKA), casein kinase I (CKI) and glycogen

synthase kinase 3β (GSK-3β) play a significant role in

regulating the Hedgehog signaling process. They all bind

to Cos2, and phosphorylate homologous domains on Ci/Gli

and Smo. Phosphorylation of Ci by PKA, CKI and GSK-3β is

shown to be essential for the efficient processing of Ci155

to its transcriptional repressor form, Ci75. Inhibition of

any of these kinases can lead to Ci155 accumulation. The

role of phosphorylation in the regulation of vertebrate Gli

proteins has not yet been clearly defined, although PKA is

shown to block vertebrate Hedgehog signaling.

roles in adult tissuesAlthough Hedgehog signaling is well studied during

embryonic development, less is known about its role

in adult tissues. Some studies have shown that Shh

activity is retained by some cells in mature organs and

dysregulation of activity in these cells, in some cases

due to mutations in Shh pathway components, leads to

tumorigenesis. Abnormal activation of the Shh pathway

has been described in a variety of human cancers and

in cancer stem cells. Loss of patched, over expression of

Shh, and activating mutations of Gli have been reported

in basal cell carcinomas, medulloblastoma, and breast

carcinomas. Loss of Smo has been linked to impaired

hematopoietic stem cell renewal and diminution in the

induction of chronic myelogenous leukemia (CML) by

Bcr-Abl. On the other hand, constitutively active Smo is

shown to cause higher proliferation of CML stem cells.

Amplification of Gli has also been shown in malignant

gliomas and osteosarcoma. Finally, mutations in Smo and

Sufu have also been associated with the formation of

sporadic basal cell carcinoma and medulloblastoma. Hence,

the Hedgehog pathway has become a potential target

for drug development for the treatment of cancers and

degenerative diseases.

references1. Merchant, A.A., and Matsui, W. Clin. Cancer Res. 2010;

16: 3130.2. Zhao, C., et al. Nature 2009; 458: 776.3. Aikin, R.A., et al. EMBO Reports 2008;9:330.4. Riobo, N.A., and Manning, D.R. Biochem. J. 2007; 403:3695. Athar, M., et al. Exp. Dermatol. 2006; 15:667.6. Jiang, J. Cell Cycle 2006; 5:2457.7. Simms-Mourtada, J., et al. Clin. Cancer Res. 2006; 12:65658. Riobo, N.A., et al. Proc. Natl. Acad. Sci. USA 2006; 103:4505.9. Macaron, N.C., et al. Arch. Dermatol. 2005; 141:259.10. Benson, R.A., et al. Mol. Immunol. 2004; 41: 715.11. Magliano, M.P., and Hebrok, M. Nat. Rev. Cancer 2003; 3:903.12. Ruizi Altaba A., et al. Nat. Rev. Cancer 2002; 2:361.13. Taylor, M.D., et al. Nat. Genet. 2002; 31:306.14. Reinferberger, J., et al. Cancer Res. 1998; 58:1798.15. Dahmane, N., et al. Nature 1997;389: 876.16. Oro. A., et al. Science 1997; 276: 817.

23

Product description Qty/Pk catalogue No.

AY 9944 Specifically blocks 7-dehydro-cholesterol reductase (Δ7-sterol reductase; DHC; IC50= 13 nM) and disrupts Shh signaling during embryogenesis.

5 mg 190080

Betulinic Acid A pentacyclic triterpene that acts as an inhibitor of Hh/Gli signaling pathway (IC50 = 32 µM).

5 mg 200498

Cyclopamine, V. californicum

A cell-permeable steroidal alkaloid and cholesterol mimic that disrupts cholesterol biosynthesis and antagonizes Shh signaling through direct interaction with Smo.

1 mg 239803

Cyclopamine-KAAD A cell-permeable Cyclopamine analog that inhibits Hh signaling with similar or lower toxicity (IC50 = 20 nM in Shh-LIGHT2 assay). Binds to SmoA1 and promotes its exit from the endoplasmic reticulum.

100 mg 239804

Hh/Gli Antagonist, GANT58

A cell-permeable blocker of downstream Hh signaling that targets Gli-mediated gene transactivation (IC50 ~5 µM in SAG-stimulated Shh-L2 cells). Reduces cellular mRNA levels of Gli1, Hip1, & Ptch, and inhibit Gli-dependent tumor growth both in vitro and in vivo.

5 mg 373400

Hh/Gli Antagonist, GANT61

A cell-permeable compound that selectively blocks downstream Hh pathway and target Gli-mediated gene transactivation (IC50 ~5 µM in SAG-stimulated Shh-L2 cells).

5 mg 373401

Hh Signaling Antagonist VII, JK184

A cell-permeable downstream Hh pathway blocker that directly targets the enzymatic activity of Adh7 (IC50 = 210 nM). Inhibits Hg-Ag-induced Gli-transcription activity (IC50 = 30 nM) as well as Gli1 and Ptc1 mRNA expression in a dose-dependent manner in H3H10T1/2 cells.

5 mg 373385

Hedgehog Antagonist VIII

A cell-permeable compound that potently inhibits OCT-Shh-stimulated Gli transcription activity in a 10t1/2(s12) cell-based Luciferase reporter assay (IC50 = 70 nM).

5 mg 373402

Hh Signaling Antagonist X, Itraconazole

A cell-permeable inhibitor of Hh signaling (IC50 ~0.8 µM against SHHN-induced Gli transcription activity in Shh-Light2 cells) that acts in a Ptch-independent manner (IC50 ~0.9 µM against Ptch promoter-mediated transcription activity in murine Ptch-/- fibroblasts). Presumably acts by binding smoothened (smo) at a site distinct from that targeted by cyclopamine and SAG.

250 mg 419825

Hh Signaling Antagonist XII, HPI-1

A cell-permeable downstream inhibitor of Hh signaling that effectively inhibits Gli-dependent transcription activity in both Sufu+/+ NIH 3T3 (shh-LIGHT2) and Sufu-/- fibroblast cultures (IC50 = 1.5 & 3 µM, respectively).

10 mg 373275

Hh Signaling Antagonist XIII, HPI-3

A cell-permeable blocker of downstream Hh signaling. Inhibits Gli-dependent transcription activity in both Sufu+/+ NIH 3T3 (shh-LIGHT2) and Sufu-/- fibroblast cultures (IC50 = 3 & 9 µM, respectively).

10 mg 373276

Hh Signaling Antagonist XIV, SANT-2

A highly potent Smo antagonist (Kd = 12 nM) that displays allosteric binding characteristics similar to SANT-1.

10 mg 373273

Hh Signaling Antagonist XV

A potent blocker of Hh-Ag1.5 to Smo (IC50 = 5 nM in CHO-K1 membrane expressing murine Smo). Blocks Gli-mediated transcription activity upon Hh-Ag1.5 stimulation in TM3-based reporter assays (IC50 = 2.7 or 35 nM against 1 or 25 nM Hh-Ag1.5, respectively).

10 mg 374274

Jervine A cell-permeable steroidal alkaloid similar to cyclopamine that induces cyclopia by blocking Shh signaling (IC50 ~500-700 nM in s12 cells).

1 mg 420210

SANT-1 A potent antagonist of Shh signaling (IC50 = 20 nM in Shh-LIGHT2 assay and in Ptch1-/- cells cells) that acts by binding to Smo (Kd = 1.2 nM). Inhibits the activities of both wild type and oncogenic Smo with equal potency (IC50 = 30 nM in SmoA1-LIGHT2 assay).

5 mg 559303

Tomatidine, HCl A steroidal alkaloid that structurally resembles cyclopamine, but lacks the capacity to inhibit Sonic Hedgehog (Shh) signaling. Useful as a negative control.

25 mg 614350

U18666A A cell-permeable amphiphilic amino-steroid that alters intracelluar membrane protein trafficking by impairing intracellular biosynthesis and transport of LDL-derived cholesterol. A weak inhibitor of Shh signaling

10 mg 662015

Veratramine, HCl, V. californicum

A cell-permeable steroidal alkaloid that is structurally related to and serves as a suitable inactive control for cyclopamine, cyclopamine-KAAD, and jervine in Shh signaling.

5 mg 676925

Available from www.millipore.com.

Sonic Hedgehog Antagonists

24

Shh Agonists

Purmorphamine, 5 mg (Qty: 5 mg, catalogue No. 540220) A cell-permeable purine compound that promotes the

differentiation of multipotent mesenchymal progenitor

cells (EC50 = 1 µM) into osteoblasts. Directly binds to and

activates Smo. Purity: ≥98% by HPLC.

Smoothened Agonist, SAG (Qty: 1 mg, catalogue No. 566660) A cell-permeable purine compound that promotes the

differentiation of multipotent mesenchymal progenitor

cells (EC50 = 1 µM) into osteoblasts. Directly binds to and

activates Smo. Purity: ≥98% by HPLC.

O

O

N

NHN

NN

N

N OCI

NH

H CIH CI

O

H3CH H

S

OH H

N

25

Hh Signaling Pathway Modulators Panel

(catalogue No. 373386) A panel consisting of 14 potent, selective, and cell-permeable antagonists, inhibitors and agonists that are useful for the

study of the Hedgehog signaling pathway. Because Hh signaling involves both intercellular and intracellular signaling,

this panel provides a convenient way to simultaneously interrogate signal-secreting and signal-receiving cells in one

experiment. The panel contains the following inhibitors and 15 mL anhydrous DMSO for reconstitution:

Nucleus

Nucleus

Hedgehog Secreting Cell

Hedgehog Receiving Cell

• GRK2 Inhibitor

• H-89, Dihydrochloride

• IC261 • H-89, Dihydrochloride

• Fluvastatin, Sodium Salt

• Smoothened Agonist, SAG • Purmorphamine

• Cyclop-KAAD• Hh Signaling Antagonist XIV, SANT-2

• IC261

• MG-132

• Hh/Gli Antagonist• GAN61

• Hh Signaling Antagonist VII, K184• Hh Signaling Antagonist XIII, HPI-3• GSK-3 Inhibitor IX, BIO• Hh Signaling Antagonist XII, HPI-1

Hh

Hh

Hedgehog

Proteosome

Secretion

Hh Precursor

-Hh

Target Genes(WNT, Ptc, BMP)Target Genes

+Hh

Gli2Degradation

N C

Cholesterol

Palmitate

N C

Ptc

N C

Ptc

N

C

Smo

SUFUGli 2/3

Gli SUFU

Microtubule

Gli

Gli 2/3GSK3β

PKA

CK1

AC

PKA

STK36

GRK CK1 GSK3β

G Proteins

GliCBPCoRCoR

GliR

GliR

components target Amount catalogue No.

Smoothened Agonist, SAG Smo agonist; Hh pathway activator 1 mg 566660

Cyclopamine-KAAD Smo antagonist 100 µg 239804

Hh Signaling Antagonist XIV, SANT-2 Smo antagonist, targets different site than Cyclopamine 10 mg 373273

Hh/Gli Antagonist, GANT61 Inhibitor of Gli binding to DNA 5 mg 373401

GSK-3 Inhibitor IX, BIO Reduces Gli1-dependent transcriptional activity 1 mg 361550

H-89, Dihydrochloride PKA Inhibitor; Hh agonist 1 mg 371963

IC261 CK1 Inhibitor (CK1δ and CK1ε; IC50 = 0.7-1.3 µM and = 0.6-1.4 µM, respectively)

5 mg 400090

GRK2 Inhibitor Reduces phosphorylation of human Smo 5 mg 182200

MG-132 Proteasomal Inhibitor 5 mg 474790

Hh Signaling Antagonist VII, JK184 Targets Adh7, inhibits Gli-transcription activity, and down-regulates the expressions of Gli1 and Ptc1

5 mg 373385

Purmorphamine Smo agonist, targets Cylopamine binding site and upregulates Gli1 and Ptc1

5 mg 540220

Fluvastatin, Sodium Salt Cholesterol biosynthesis inhibitor 25 mg 344095

Hh Signaling Antagonist XII, HPI-1 Remains effective against Sufu-/- fibroblasts overexpressing Gli1 or Gli2

10 mg 373275

Hh Signaling Antagonist XIII, HPI-3 Ineffective against Sufu-/- fibroblasts over-expressing Gli1 or Gli2

10 mg 373276

DMSO - 15 mL KP31817

Phosphorylation

Ubiquitin

GTP

Calbiochem® Inhibitors

26

Immunohistochemistry of paraffin-embedded kidney tissue using Anti-Protein patched homolog 1 shows that expression is restricted to proximal tubules with no distal tubule involvement. Immunostaining was performed using a 1:300 dilution of catalogue No. 06-1102, Anti-Protein patched homolog 1. reactivity was detected using the IHc-Select detection Kit (catalogue No. dAB050).

Immunohistochemistry using a 1:500 dilution of catalogue No. 06-1106, Anti-Sonic Hedgehog was performed on a section of paraffin-embedded colorectal carcinoma tissue. reactivity was detected using the IHc Select® detection Kit (catalogue No. dAB050).

HeLa cell lysate probed with Anti-Protein patched homolog 2 (0.5 µg/mL). Proteins were visualized using a donkey Anti-rabbit IgG conjugated to horseradish peroxidase and a chemiluminescence detection system. Arrow indicates Protein patched homolog 2 (~130 kda).

Antibodies for Sonic Hedgehog Signaling research

Anti-Protein patched homolog 1 (Qty: 100 µg, catalogue No. 06-1102)

Hedgehog signaling is regulated by its receptor, Protein

patched homolog 1, which keeps the pathway turned off

in the absences of activation. The immunogen used to

develop this purified rabbit polyclonal antibody was a KLH-

conjugated linear peptide corresponding to the cytoplasmic

domain of human Protein patched homolog 1. Recognizes

the cytoplasmic domain of patched homolog 1 in human

and mouse. Applications: Western blot, IHC Anti-Sonic Hedgehog, affinity-purified rabbit polyclonal (Qty: 100 µL, catalogue No. 06-1106)

Immunogen used was GST-tagged recombinant protein

corresponding to the N-terminus of Sonic Hedgehog.

Recognizes the N-terminus of Sonic Hedgehog in human

and mouse. Applications: Western blot, IHC

Anti-Smo Mouse mAb (1d9) (Qty: 100 µg, catalogue No. St1718)

Immunogen used was a full-length, recombinant, human

Smo (aa 653-787) expressed as a GST fusion protein.

Recognizes 75 kDa Smo in A431 cells.

Applications: Western blot

Anti-Sonic Hedgehog (c-product), clone EP1190Y, rabbit monoclonal (Qty: 100 µL, catalogue No. 04-971)

Immunogen used was a synthetic peptide corresponding

to residues within the c-product subunit of human Shh

protein. Recognizes both full-length (50 kDa) and c-product

subunit (27 kDa) of human Shh protein. Applications: Flow

cytometry, Western blot, IHC (paraffin), IP.

Anti-Protein patched homolog 2 (Qty: 100 µg, catalogue No. 06-1103)

Protein patched homolog 2 and Smoothened form a

membrane complex that serves as a receptor for Shh.

The immunogen used to develop this purified rabbit

polyclonal antibody was a KLH-conjugated linear peptide

corresponding to human Protein patched homolog 2 at

and around the extracellular domain. Recognizes Protein

patched homolog 2 in human, canine, equine, and mouse.

Applications: Western blot, IHC

260(kDa)

160

110

0

60

5040

3020

1510

27

Product HIGHLIGHtProduct HIGHLIGHt

Shaping Epigenetics Discovery. Chromatin, DNA methylation, RNA analysis.

With EMD Millipore, epigenetics technology has never been more accessible or easier to use. From highly specific antibodies, to rapid bisulfite kits, to industry leading ChIp and next generation sequencing kits, we simplify epigenetics applications to allow you to design the most ambitious experiments to interrogate gene regulation.

With our legacy of expertise from Chemicon® and Upstate®, we are committed to molding sophisticated yet simple solutions for understanding epigenetic regulation.

To learn more visit:

www.millipore.com/EPIc

EMD Millipore, the M mark, Muse, LentiBrite, Magna ChIP, ChIPAb+ and Magna GrIP are trademarks and Chemicon, Upstate, Calbiochem, guava, EndoGRO, IHC Select, MILLIPLEX, Millicell are registered trademarks of Merck KGaA, Darmstadt, Germany. Trademarks belonging to third parties are the properties of their respective owners. Lit. No. PR4888EN00 07/12 Printed in U.S.A. LS SBU-12-06723© 2012 EMD Millipore Corporation, Billerica, MA 01821 U.S.A. All rights reserved.

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Focused on your research: Your partner for epigenetics, cell signaling, cancer, neuroscience, toxicity, cell structure and stem cells, EMD Millipore provides you with the antibodies and small molecules you need most.

Validated: Generate reproducible results with antibodies that undergo rigorous quality control testing and are validated Western blotting, immunoprecipitation, immunohistochemistry, ELISA, flow cytometry and more. Our small molecules are well-characterized and documented (including solubility data) to ensure that you get the best results.

100% Guaranteed: Relax. EMD Millipore antibodies and small molecules are backed by our best-in-industry technical support. With a 100% Performance Guarantee, what do you have to lose? If you are not completely satisfied with the performance of EMD Millipore antibodies or small molecules, contact a technical support specialist for assistance or full credit* against future purchases.

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*EMD Millipore’s Antibody Guarantee: Valid one year from date of receipt. Does not apply to bulk, custom or commercial order. Subject to change without notice, and applies to validated antibody applications only.

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