Additional file 1 of SMAD1 as a biomarker and potential therapeutic target in drug-resistant multiple myeloma
Creators: name: Wu, Jian
name: Zhang, Min
name: Faruq, Omar
name: Zacksenhaus, Eldad
name: Chen, Wenming
name: Liu, Aijun
name: Chang, Hong
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Hematology

description: Additional file 1: Supplementary materials and methods. Fig. S1. Downregulated SMAD1 reduced migratory and invasive abilities of MM cells in vitro (A) Representative images of soft agar colony formation assays with the different cell models. Bar graphs indicate the colony count expressed in different microscopic fields. (B) Migration ability toward serum of 8226R5 and OPM2 vel/R cell lines were assessed using Transwell filter. Cells were pre-treated with DM 5uM for 24 h prior to the assay. (C) Migration ability toward serum of 8226R5 and OPM2 vel/R cell lines were assessed using Transwell filter. Cells were pre-treated with siSMAD1 50 nM and/or TGF-β 24 h Data were presented as mean ± s.d. from at least separate experiment. *: p &lt; 0.05; **:p &lt; 0.01; ***:p,0.001; Fig. S2. Correlation of SMAD1 expression with apoptosis activity (A) Correlation analysis of SMAD1 expression on the y axis and the Z-score enrichment across over 1000 cell lines from CCLE database. A Z-score was generated for each cell line in KEGG canonical pathway gene sets: apoptosis. Red circles indicate MM cell lines, and black circles indicate all CCLE cell lines except MM. A significant correlation between SMAD1 expression and the apoptosis activation was observed; R = − 0.1486, p &lt; 0.0001.(B-F) Correlation between SMAD1 expression and apoptosis activation was also observed in other hematological malignancies. Fig. S3. SMAD1 inhibition induces apoptosis in MM cells. (A) OPM2 vel/R, MM1.R cell lines were treated with 10uM of either DM or DMSO control for 24 h, treated with 10 nM BTZ or vehicle for 48 h, and then subjected to annexin-V/propidium iodide analysis by flow cytometry to determine percentage of apoptotic cells(left). (B) OPM2 vel/R, MM1.R cells lines were transfected with 30 nM of either siSMAD1 or siRNA control for 24 h, treated with 10 nM BTZ or vehicle for 48 h, and subjected to annexin-V/propidium iodide analysis to determine percentage of apoptotic cells. Fig. S4. Combination of drugs (Dox, Dex, Len, DM) synergistically induces cytotoxic effects on primary MM patients’ sample. (A) Primary mononuclear cells derived from 5 MM patients were treated with indicated concentration of Dox, Dex, Len and DM for 48 h, and then cell viabiility was evaluated by MTT assays. (B) CD138 + cells derived from 2 MM patients were treated with indicated concentration of Dex, Dox, Len and DM for 48 h, and then cell viabiility was evaluated by MTT assays. (C) PBMCs derived from three healthy donors were treated with indicated concentrations of DM alone or in the presence of 10 nM BTZ for 48 h and cytotoxicity was assessed by MTT. Results are presented as mean ± s.d. from at least separate experiment. *: p &lt; 0.05; **:p &lt; 0.01; ***:p,0.001; NS: not significant. Fig. S5 (A)SMAD1 binding sties and histone modification around the NF-κB1 promoter identified by Chip-seq data from ENCODE. (B) Genome-wide identification of transcription binding sites of SMAD1 in the NF-κB1 promoter region based on UCSC data. (C) 16 putative sites were predicted with these setting (80%) in sequence named hg38_genscan_chr4.1488. Fig. S6. Representative microscopic images of tumor sections from four treated groups analyzed for histology (H&amp;E), proliferation (Ki-67) or apoptosis (TUNEL). STable 1. The top 5 KEGG pathway enriched by GSEA in APEX trial dataset by comparing the SMAD1-high and SMAD1-low expression samples. Fig. S7 (A) In parallel with cell cycle assay, western blot was performed to measure the efficiency of siRNA in MM cell lines. (B) In parallel with immunofluorescence, Protein lysate was subjected to western blot with indicated antibodies.