Research Paper Volume 9, Issue 3 pp 860—879

Figure 6. Silencing of DIRAS3 in ASCs induces both premature senescence and adipogenic differentiation. (A) (Left panel) Adipogenic differentiation of ASCs was estimated by staining the cells with Oil-Red-O stain at day 9 post induction of differentiation. Non-induced ASC controls are shown. (Right panel) Quantification of Oil-Red-O stained area using image J is shown (n=3). (B) Expression of PPARγ2, FABP4 and Perilipin mRNA was estimated at day 3 and day 9 post induction of adipogenesis. Expression at day 0 before induction was taken as 1 and fold increase was calculated. Values are normalized to β Actin. (C) Perilipin and p16^INK4A protein levels were analysed in DIRAS3 KD and control ASCs at day 9 post induction of adipogenesis by western blotting. β-Actin protein served as input control. (D and E) Densitometric evaluation of western blots bands from figure C. Fold changes in densitometric band intensities of perilipin (D) and p16^INK4A (E) normalized to β-Actin protein, acquired by image J were plotted. (F) p16^INK4A mRNA expression was analysed in DIRAS3 KD (red) and control ASCs (black) at day 3 and 9 post adipogenesis induction by q-RT_PCR analysis. All error bars represents the means ± SEM. p values * = p<0.05, **= p<0.001 and *** = p<.0001.