Figure 2. Silencing of DIRAS3 reduces proliferation and self-renewal of human ASCs. (A) Proliferation of ASCs following knock-down (KD) of DIRAS3 was monitored by cell counting (n=4). (B and C) Monitoring of cell proliferation by CFSE signal dilution technique. Flow cytometric analyses demonstrated a higher proliferation rate of shCntrl relative to shDIRAS3 infected ASCs, reflected by a significantly higher percentage of CFSE low cells compared to shDIRAS3 infected ASCs. Differentially transduced ASCs were stained with CFSE and cultured for 4 days. Dilution of CFSE signal was monitored by FACS (B). Percentage of CFSE low cells was plotted (C) (n=4). (D and E) ASCs were seeded at a density of 500 cells in tissue culture dish following transduction with shDIRAS3 and shCntrl expressing lentiviruses and selection. Number of colonies were counted 10 days post seeding after staining with crystal violet (D) and plotted (E) (n=3) (F) Expression of Ki-67 was assessed in transduced ASCs via confocal microscopy. Cells were fixed on cover slips and stained for Ki-67 (green) and DAPI (blue) for nuclear staining (n=3). All error bars represents the means ± SEM. p values *** = p<.0001.