Research Paper Volume 9, Issue 3 pp 860—879

Figure 1. DIRAS3 knock-down (KD) leads to an upregulation of Akt-mTOR signalling in proliferating ASCs. (A) Efficiency of DIRAS3 KD in ASCs. DIRAS3 mRNA levels were measured by quantitative real time PCR (q-RT PCR) following infection of ASCs with lentiviruses expressing DIRAS3 specific shRNA. An empty vector control (shEmpty) and a scrambled sh sequence (shCntrl) are employed as controls (n=4). (B) Activation of Akt and p70S6K by DIRAS3 KD in ASCs. (Left panel) DIRAS3 was KD in ASCs using specific shRNAs as indicated. Cells were starved by serum withdrawal for 48 hours. Afterwards cell lysates were prepared and phosphorylation of Akt (S473) and P70S6K (T389) was examined by immune-blotting using specific antibodies. GAPDH served as an input control. (Right panels) Fold changes in densitometric band intensities presented as Arbitrary Units (AU) for phosphorylated proteins normalized to un-phosphorylated total proteins, acquired by image J were compared. Band intensity of shCntrl 0% FCS 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.