nature | methods

SUnSET, a nonradioactive method to monitor protein synthesis

Enrico K Schmidt, Giovanna Clavarino, Maurizio Ceppi & Philippe Pierre

Supplementary figures and text:

Supplementary Figure 1 Monitoring translation by immunoblot.

Supplementary Figure 2 Puromycin pulses do not impact on other cellular functions.

Supplementary Figure 3 Monitoring translation by confocal microscopy.

Supplementary Figure 4 SUnSET monitoring and 35S incorporation in primary DCs.

Supplementary Figure 5 SUnSET monitoring of anti-proliferative drug activity.

Supplementary Methods

Nature Methods: doi: 10.1038/nmeth.1314

Nature Methods: doi: 10.1038/nmeth.1314

Nature Methods: doi: 10.1038/nmeth.1314

Nature Methods: doi: 10.1038/nmeth.1314

Nature Methods: doi: 10.1038/nmeth.1314

Nature Methods: doi: 10.1038/nmeth.1314

Supplementary Methods

T cell activation. 5 µg/ml SL8 (SIINFEKL) peptide (Schafer-N, Denmark) were

added to DCs for 4 h. After harvesting, DCs were fixed with PBS/ 0,25 % PFA for 5

min at RT. After washing, PFA was quenched with 10 mM glycin for 5 min at RT.

20.000 DCs were mixed with 200.000 T cells and incubated in complete T cell

medium at 37 °C and 5 % CO2. T cell receptor (TCR) activation was analysed by

FACS with anti-CD69 antibody (BD Biosciences) and T cell activation/proliferation

with the CellTiter-Glo Luminescent Cell Viability Assay (Promega). As positive control

for TCR activation T cells were treated with 5 µg anti CD3 antibody.

SUnSET. We performed all experiments in 96 round bottom well plates (Greiner bio-

one) with maximal cell number of 200.000/ 150 µl RPMI complete medium (RC)

including 10 % FCS (HyClone Perbio) and 100 units/ml penicillin/streptomycin

(Gibco). All centrifugations are done for 3 min and 340 g. 15 µl of 10 µg/ml puromycin

solution (SIGMA, min. 98 % TLC, cell culture tested, P8833, diluted in PBS) were

added and cells incubated for 10 min at 37 °C and 5 % CO2 (= pulse). Cells were

centrifuged at room temperature (RT), two times washed with 150 µl prewarmed RC

using the same centrifugation setup, resuspended in 150 µl prewarmed RC and

incubated for 50 min at 37 °C and 5 % CO2 (= chase). Cells were harvested by

centrifugation at 4 °C and two times washed with cold PBS including 0,1 % BSA

(PBS/BSA). Cells were resuspended in 30 µl cold PBS/ BSA including 1 µg A647-

labelled mouse IgG2a anti-puromycin antibody (12D10) and incubated 30 min on ice.

Cells were two times washed with 150 µl PBS/ BSA and fixed by re-suspending in 40

µl 2 % paraformaldehyde (PFA )solution (diluted in PBS/BSA) and stored at 4 °C until

FACS analysis.

Immunoblotting and radioactive labeling. 50 µg of TX-100 soluble material or

200.000 cells, lysed directly in Laemmli, were loaded on SDS-PAGE (2 to 12%

gradient or 10 % non-gradient) prior immunoblotting and chemiluminescence

detection (Pierce, USA). The mouse monoclonal anti-actin antibody (clone AC-15)

was purshased from Sigma and the rabbit polyclonals anti-phospho-S6 (Ser235/236),

Nature Methods: doi: 10.1038/nmeth.1314

anti-phospho-p70S6K (Thr389) and anti-phospho-eIF2! were from Cell Signaling

Technologies. anti-P62 and anti-eIF2! antibodies was from Santa Cruz.

2 x 107 cells were pulse-labelled with 10 mCi/ml of [35S]-methionine Pro-mix (APB,

UK) for 10 min at 37 °C in RPMI/ 10 % FCS prior SDS-PAGE separation and

quantification with a FUJI phosphorimager. In addition, TX-100 soluble material from

200.000 cells were incubated in 25% TCA (6,1N) for 10 min at 4°C to precipitate

proteins. After 5 min centrifugation at 16.000 x g the pellet was washed with ice cold

acetone. The dryed pellet was resuspended in PBS and cpm were determined by

liquid scintilation counting (LSC).

Flow cytometric analysis. Cells were stained with specific for cell surface markers

CD8a (RM2201, Caltag), CD69 (H1.2F3), CD45.2 (104, both BD Pharmingen),

CD42.1 (A20), CD11c (N418, both eBioscience) or puromycin (12D10) 30 min at 4

°C. Cells were then washed and fixed in 2% paraformaldehyde in PBS. Events were

collected on a FACScalibur and the data were acquired using CellQuest software (BD

Biosciences) and analyzed using FlowJo.

Polysomes separation by sucrose gradient fractionation. 20x106 cells were lysed

in 1 ml of polysome buffer (10 mM Tris-HCl (pH 8), 140 mM NaCl, 1.5 mM MgCl2,

0.5% NP40, 0.1 mg/ml cycloheximide, and 500 units/mL RNasin (Promega,

Madison, WI). After 10 min on ice, lysates were quickly centrifuged (10.000 x g for 10

sec at 4°C) and the supernatant was resuspended in a stabilizing solution (0.2 mg/ml

cycloheximide, 0.7 mg/ml heparin, 1 mM phenylmethanesulfonyl fluoride). After a

quick centrifugation (12.000 x g for 2 min at 4°C) to remove mitochondria and

membrane debris, the resulting supernatant was layered on a 15% to 40% sucrose

gradient. Gradients were then ultracentrifuged (35.000 x g for 2 h at 4°C) and after

centrifugation 20x 1 ml fractions were collected, starting from the top of the gradient.

The correct fractionation of the polysomes was tested by detecting the different rRNA

types on a 1% denaturing agarose gel. The intensity of the 18S and 28S rRNA bands

was quantified using the software ImageQuant (Fuji).

Nature Methods: doi: 10.1038/nmeth.1314

Mice. Male C57BL/6 mice 7-8 weeks old (Charles River Laboratories) were

purchased for use in these studies. Ovalbumin-specific T-cell receptor expressing

mice (OT-1) were bred in pathogen-free breeding facilities at the CIML (Marseille,

France). Experiments were conducted in accordance with institutional guidelines for

animal care and use. Protocols have been approved by the French Provence ethical

committee (number 04/2005).

Preparation of anti-puromycin antibodies. Puromycin (Sigma) was covalently

attached to keyhole limpet hemocyanin (KHL, Calbiochem 374817). The final

reaction volume was 3 ml with 5 mg/ml KHL, 3,5 mM puromycin, 200 mM

sodiumphosphate pH 7,5, 57,5 mg 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

(EDC) and 5 mM N-hydroxysulfosuccinimide (Sulfo-NHS, PIERCE 24510). The

reaction was started by adding solid EDC, incubated for 30 min at 22°C and stopped

on ice by adding 50 mM Tris pH 7,4. The reaction was exhaustively dialyzed against

10 mM potassium phosphate pH 7,4 and 144 mM NaCl. The immunisation, fusion,

cloning and screening by ELISA was performed by EMBL Monoclonal Core Facility,

(Monterotondo-Scala, Italy). After subcloning two isotypes could be raised, IgG2a"

(12D10) and IgG1" (2G11), and validated for Western blot, immunofluorescence

microscopy, FACS and immunoprecipitation. For FACS analysis, the 12D10 was

directly labelled with fluorochrome A647 using Alexa Fluor# 647 Protein Labelling Kit

(A20173, Molecular Probes).

Cell culture. NIH3T3, JURKAT and B3Z cells were maintained in RPMI 1640

(GIBCO BRL Life Technologies, Rockville, MD) supplemented with 10% foetal calf

serum (FCS, HyClone, PERBIO, Aalst, Belgium), at 37°C and 5% CO2. Bone

marrow-derived DCs were obtained and cultured as described previously {Lelouard,

2007 #1880}. Maturation of DCs was induced using 100ng/ml LPS. For isolation of

CD8! T-cell populations, spleens from OT-1 SL8-specific TCR transgenic mice were

digested by collagenase (liberase CI; Roche 11814435001), teased apart by

Nature Methods: doi: 10.1038/nmeth.1314

repeating pipetting in PBS, 5 mM EDTA, and 2% FCS (PBS/EDTA/FCS), passed

through nylon mesh and washed. T-Lymphocytes were separated using CD8a+ T Cell

Isolation Kit and protocoll von Miltenyi Biotec (130-090-859).

Surface protein isolation. Surface protein were biotinylated, cells lysed and labelled

protein isolated according to the instructions of the Cell Surface Protein Isolation Kit

(PIERCE). Isolated proteins were analysed by immunoblot with anti-puromycin

12D10.

Immunocytochemistry and mRNA in situ hybridization. Cells were treated with

25 !M cycloheximide (Sigma) for 5 min or 500 !m sodium arsenite (Sigma) for 30

min, then 10 µg/ml puromycin (Sigma) was added for 10 min in the culture medium.

Cells were then fixed in 3 % PFA in PBS for 15 min at RT, permeabilized with 0,1 %

saponin in PBS/ 5 % FCS/ 100 mM glycine for 15 min at RT and stained 1 h with

primary antibodies anti-puromycin 12D10 and anti-phospho-eIF2! (rabbit polyclonal

phospho-specific from Biosource). All Alexa secondary antibodies were from

Molecular Probes, Inc (Eugene, OR). Stress granule formation was detected by in

situ hybridization with oligo-dT (Alexa Fluor 555 dT18, Invitrogen). In this case, cells

were fixed with PFA, permeabilized with methanol 10 min at –20 °C and incubated

with oligo-dT for 4 h at 43 °C. Next, stainings with the primary and secondary

antibodies were performed. Immunofluorescence and confocal microscopy (using

microscope model LSM 510; Carl Zeiss MicroImaging, Inc.) were performed as

described previously Lelouard et al, 2004. The profiles of intensity of fluorescence of

phospho-eIF2! and puromycin were analysed using the LSM Zeiss Image software.

Nature Methods: doi: 10.1038/nmeth.1314