Figure 3E. (Continues). Silencing of DIRAS3 induces premature senescence in human ASCs. DIRAS3 was KD in ASCs using lentiviruses expressing specific shRNA at increasing MOI. Cell lysates were immunoblotted with the specific antibodies to investigate accumulation of senescent associated proteins p16INK4A, p21CIP1, p53 phosphorylated p53 (S15), Rb and pRb (S807/811). β-Actin served as a loading control. Fold changes in densitometric band intensities for phosphorylated proteins normalized to un-phosphorylated total proteins, acquired by image J were compared. Band intensity of shCntrl was taken as 1. Western blot shown is from replicate from one donor with similar protein expression pattern was observed with 2 different donors. All error bars represents the means ± SEM. p values * = p<0.05, **= p<0.001 and *** = p<.0001.