Fig. 4

FAM46C control survival and proliferation of multiple myeloma cells. a–c Expression of FAM46C ^WT induces death in multiple myeloma cells. a An example gating strategy for defining transduction efficiency and cell viability. Forward scatter (FSC) and side scatter (SSC) gate were used to separate debris from intact cells. Viability of RPMI8226 cells overexpressing either FAM46C^WT-GFP or FAM46C^mut-GFP was analyzed using propidium iodide (PI) staining on the 11th day post-transduction. GFP expression (GFP+) was evaluated in parallel in PI-negative cells. b, c Summary of flow cytometry analyses presented as bar graphs showing GFP expression level and reduced viability of multiple myeloma cell lines (SKMM1, H929, and RPMI8226 in b and Raji and HL60 in c throughout the time course of GFP, FAM46C^WT-GFP, and FAM46C^mut-GFP expression. The data are presented as percentage of cells ± SD (n = 3). P values were calculated using two-way ANOVA tests (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). d, e shRNA-mediated silencing of FAM46C enhances proliferation rate of RPMI8226 MM cell line expressing wild-type protein but not SKMM1 harboring FAM46C mutation. Cells were transduced with lentiviral vectors expressing shRNA and empty vector as control. Stable transduced cell lines were stained with CFSE. Cell division was monitored by flow cytometry after 48 and 72 h by levels of CFSE dilution. d Reverse transcription qPCR analysis of the FAM46C silencing efficiency. Bars represent mean values ± SD (n = 3). e The rate of proliferation of shRNA treated cells normalized to the control transduction. Bars represent mean values ± SD. P-values were calculated using Student’s t-test (*P < 0.05), (n = 3)