Supplementary material and methodsDrugs and reagents

Bortezomib, Selleck Chemicals, Houston TX 77230 USA;

MPS, Synpeptide Co, Shanghai, China

MPS peptide sequences:

MPS peptide: KKKKKRFSFKKSFKLSGFSFKKNKK

Mutated MPS peptide: KKKKKRFDFKKDFKLDGFDFKKNKK

Antibodies for IP and Western blotting:

Anti-Marcks, Cell Signaling Technology, Inc. Danvers, MA

Anti-pMarcks, Cell Signaling Technology, Inc. Danvers, MA

Anti-bacline 1, Cell Signaling Technology, Inc. Danvers, MA

Anti-PUMA, Cell Signaling Technology, Inc. Danvers, MA

Anti-LC3B, Cell Signaling Technology, Inc. Danvers, MA

Anti-GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), Cell Signaling Technology, Inc.

Danvers, MA

Anti-β-Actin, Sigma-Aldrich, Saint Louis, Missouri 63103 USA

Immunoprecipitation and immunoblotting

Cells used for immunoprecipitation were re-suspended in pre-cooled modified Triton lysis buffer

(1% Triton X-100, 20mM Tris-HCl with pH 7.4, 2.5mM EDTA, 2.5mM ethylene glycol

tetraacetic acid, 140mM NaCl, 2 µM β-Me, 1 mM PMSF and cocktail inhibitors tablets (Roche))

and then incubated 30 min on ice. Protein extracts were harvest by centrifuging the cell lysate in

a refrigerated centrifuge and then incubated overnight at 4°C with indicated antibodies or

negative control IgG. Antibody-bonded target proteins were immunoprecipitated with protein G

beads and analyzed on 10-15% SDS-PAGE. The membranes were probed with indicated

antibodies and the bands were visualized using HRP-conjugated secondary antibody and ECL

substrate (Thermo).

Quantitative real-time PCR

Total RNA including miRNA was extracted using miRNeasy mini kit (Qiagen). cDNA was

synthesized by miScript II RT Kit (Qiagen), and applied to miRNA real time PCR using miScript

SYBR Green PCR Kit (Qiagen) according to the manufacturer's instructions. The expression of

mature miRNAs was calculated relative to SNORD72 and fold changes in miRNA treatments

relative to scrambled treatments were calculated by using the 2ΔΔCt algorithm. The mRNA

expression of target genes was determined by RT-qPCR for MM cell lines. Briefly, total RNA

was extracted using TRIzol reagent (Invitrogen). cDNA synthesis and quantitative RT-PCR

(qRT-PCR) were performed according to the manufacturer’s instructions (Biorad). Ct values

were extracted and after normalization to GAPDH and other housekeeping genes, fold changes

in gene expression were determined using the 2-ΔΔCt algorithm.

Gene silencing with transfection and transduction

MM cells were transiently transfected with either siMARCKS or a scrambled siRNA (Ambion)

using Lipo3000 Transfection Reagent (life) according to the manufacturer’s instructions. Assays

were performed after 48 hrs incubation at 37℃ and 5% CO2. Transduction of MARCKS

silencing was accomplished using Lentiviral shRNA particles. 1.0×106/mL HEK 293T cells

were seeded in complete medium and were incubated 24hrs at 37℃ and 5% CO2. Then cells

were transfected with packaging plasmids (pMDlg/pRSV/pMD2) and either shMARCKS or

scrambled shRNAs coded plasmids by using Lipo3000 Transfection Reagent according to the

manufacturer’s instructions. Cells were continuously incubated for 48 hours and the medium

containing virus particles was harvested and filtrated with 0.45 µm filters (Millipore) if the

florescence signals of the cell are visible. To transduction the MM cells, 5.0×106 MM cells were

re-suspended in 3 mL lentivirus particles containing medium with 8 μg/mL PolyBrene®, and

then centrifuged for 2hrs at 3200 rpm. Re-suspended the cells carefully with fresh RPMI 1640

medium and further incubated 48 hrs to check the florescence signals. Cells constantly

expressing the shRNA-coding genes were screened with RPMI 1640 medium containing 2.0

μg/mL puromycin and maintained in culture with selection media until use.

Cell viability and apoptosis assay

Cell viability was assessed by MTS colorimetric assay. MM cell lines or PBMCs obtained from

healthy donors were seeded in 96-well plates in 100-μL complete medium at a density of 2.0 ×

104 cells per well. MM cells were incubated at 37℃ and 5% CO2 with various concentrations of

indicated compounds or peptides for indicated periods. After the incubation, 10 µl of MTS (0.5

mg/mL) was added, and the cells were further incubated for an additional 3 hours away from

light at 37℃, followed by adding acidified isopropanol to each well. The absorbance was read

with a microplate reader set at a test wave length of 570 nm and a reference wavelength of 630

nm. Each experiment was performed in triplicate and the mean value and SD were calculated.

Each experiment was performed in triplicate and the mean value and SD were calculated. To

examine apoptotic cell death, MM cells were treated with various concentrations of BTZ in

combination with miR-34a for 48 h followed by annexin V-FITC/PI staining for apoptosis

analysis in a FACS Calibur flow cytometer (Becton Dickinson). Captured events were analyzed

using CellQuest software. The extent of apoptosis was quantified as percentage of annexin-V

positive cells, and the extent of drug-specific apoptosis was assessed by the formula: percentage

of annexin-V positive cells = (test - control) × 100/ (100 - control).

Acridine orange staining

Cells used for AO staining were seeded in 12-well plates and stained with 5 µg/mL acridine

orange for 15 min. Surplus acridine orange was washed by PBS and cells were observed under

fluorescence microscope following re-suspended in fresh complete medium.

Supplementary Figures

Supplementary figure S1: Targetscan analysis for MARCKS-3’UTR. (A) Schema of the

candidate miRNAs by different TargetScan algorithms. Each labelled circle represents one target

prediction algorithm with the number of its predicted miRNAs. The number of miRNAs,

predicted by two, is listed in overlapping area of circles. The 9 common miRNAs in two

different targetscan Algorithms with the highest target score presented in the figure. (B)

Correlation analysis of endogenous 8 miRNAs (other than miR-34a with high score for targeting

prediction Algorithms) with MARCKS expression in patient dataset (GSE70319) presented as

scatter plots. (C) The box whisker plot showing miR-34a and 8 miRNAs expression in

GSE17498 dataset (stage I (durie-salmon): n=9, stage II (durie-salmon): n=9 and stage III (durie-

salmon): n=12). (D) GSEA analysis of an enrichment the miR-34a target genes in MM patients

who display a high level of MARCKS expression in two MM datasets (GSE2658 and

GSE17306). (E) The 8226R5 and MM.1R transduced cells with miR-34a or scramble control

lenti vectors were treated with 10 nM BTZ or vehicle for 48h. Then the cells were stained with

annexin-V/propidium iodide and analyzed by flow cytometry to determine the percentage of

apoptotic cells. The percentage of double positive cells indicated in each dot plot as a

representation of apoptosis. (F) The western blot analysis represents the MARCKS protein level

in RPMI-8226R5 and MM.1R transduced cell lines with shMARCKS or scramble control. (G)

The miR-34a level was assessed in the shMARCKS-transduced RPMI-8226R5 and MM.1R cells

with shMARCKS or scramble control by qPCR. The expression level was normalized to

SNORD72 gene. Results shown are mean±SEM.*, P < 0.05; **, P < 0.01; and ***, P < 0.001 (n)

Supplementary figure S2: (A) whole cell lysate was prepared from pair MM cells and subjected

to WB. P-MARCKS expression was determined by western blot analysis in RPMI-8226 vs.

RPMI-8226R5 and MM.1S vs MM.1R cells, respectively. (B) The RPMI-8226R5 and MM.1R

shMARCKS -transduced cell lines were treated for 48 hrs with indicated concentrations of BTZ

and the cell viability was measured by MTT. (C) Median-effect plot of 48hrs MPS-bortezomib

co-treatment. Median effect was calculated by CompuSyn software. (D) The RPMI-8226R5 and

MM.1S cell lines were treated for 48 hrs with indicated concentrations of MPS and BTZ

following by measuring the cell viability by MTT.

Supplementary figure S3: (A) Tumors treated as above were analyzed by immunoblotting for

p-MARCKS, LC3B and GAPDH. (B) Representative Images of tumor tissues slides from five

treated mice groups.

Supplementary figure S4: (A) Western blot analysis was performed to determine the expression

levels MARCKS and p62 in multiple myeloma cell lines MM.1R treated with 40µM MPS or

Mut-MPS (left). Quantification of protein levels of MARCKS and p62 measured by Image J

(right). Data are means±Standard Error of Mean. *P≤0.05, **P≤0.01, ***P≤0.001. (B)

Representative confocal images showing LC3B-positive vesicles in 8226R5 multiple myeloma

cells treated as indicated. LC3B expression is shown on the left panel. Middle panel is the

overlay of LC3B expression and DAPI staining (nuclear staining). Right panel shows the 3D

representations after deconvolution of the 488nm signal, which represents the LC3B expression.

(C) Quantification of the number of LC3B-positive vesicles in Fig. S4B after 3D deconvolution

of images (upper panel), and quantification of the number of LC3B-positive vesicles multiplied

by the volume of these vesicles after 3D deconvolution of images (lower panel). (D)

Representative images of acridine orange staining of shMARCKS -transduced RPMI-8226R5

and MM1R cell lines or control cells. (E) Western blot analysis was performed to determine the

phosphorylation level (T198) in MARCKS silenced MM cells. (F) Western blot analysis was

performed to check the LC3B and PUMA level in MM1.S and MM1.R cells after 24 hours MPS

or Mut-MPS treatment with indicated concentrations.

Supplementary figure S5: shMARCKS-transduced RPMI-8226R5 and MM1R cell lines or

control cells were treated with BTZ (5nM) and different indicated concentrations of CQ and the

cell viability was measured with MTT assay.

Supplementary figure S6: (A) The MM.1R and 8226R5 cells were treated with different

concentration of MPS or mut-MPS as a negative control for 24hrs and the level of PUMA was

assessed by western blot. (B, C) The protein lysate was prepared from shMARCKS -transduced

MM1R/RPMI-8226R5 cell lines or control cells and immunoprecipitated with either baclin-1 or

PUMA antibodies. The Input and IP samples were subjected to western blot by indicated

antibodies. Possessions of Bcl-xL and Mcl-1 by PUMA are dampened in 8226R5/MM1R cells

transfected with shRNA targeting MARCKS. Beclin-1 is released from Bcl-xL in MARCKS

silenced 8226R5 cells.

Table S1. Synergistic effects of CQ and BTZ co-treatment are more significant in MARCKS

silenced MM1R and 8226R5 resistant MM cell lines

CI Data for Non-Constant Combo on 8226R5 sc: C&B (CQ+BTZ)

CI Data for Non-Constant Combo on MM1.R sc: C&B (CQ+BTZ)

Dose BTZ (nM)

Dose CQ (µM)

Cell Viability %

CI Dose BTZ (nM)

Dose CQ (μM)

Cell Viability %

CI

5.0      40.0      43.61 0.7 7.5      40.0      19.36      0.71

5.0      60.0      41.9 0.72 7.5      60.0      16.92      0.81

5.0      80.0      37.19 0.65 7.5      80.0      16.26      0.95

       

CI Data for Non-Constant Combo on 8226R5 shMARCKS: C&B (CQ+BTZ)

CI Data for Non-Constant Combo on MM1.R shMARCKS: C&B (CQ+BTZ)

Dose BTZ (μM)

Dose CQ (μM )

Cell Viability %

CI Dose BTZ (μM)

Dose CQ (nM)

Cell Viability %

CI

5.0      40.0      37.57      0.57 7.5      40.0      19.36      0.64

5.0      60.0      29.11      0.38 7.5      60.0      16.92      0.72

5.0      80.0      25.92      0.33 7.5      80.0      16.26      0.85