Fig. 3.

DIRAS3 is a negative regulator of adipogenesis in human ASCs. (A) DIRAS3 mRNA expression normalized to actin was investigated by q-RT-PCR during the course of adipogenesis (n = 6). (B) Akt–mTOR pathway activity pattern was monitored during the first 72 h of adipogenesis upon DIRAS3 KD. Phosphorylation of Akt (S473) and S6K1 (T389) was examined by Western blotting. (C) ASCs infected with either shCntrl or shDIRAS3 expressing lentiviruses were subjected to adipogenesis and mRNA expression of adipocyte marker genes FABP4, Perilipin and Adiponectin normalized to actin were analyzed using q-RT-PCR at the indicated time points. (D) Perilipin protein expression was analyzed by western blotting at day 9 post-adipogenesis induction in shCntrl and shDIRAS3 ASCs. (E) Adipocyte differentiation was estimated using Oil-Red-O staining at day 9 post-induction. (F and G) Representative immunohistochemical staining of xenotransplanted shDIRAS3 SCID mice (F) and shCntrl mice (G) using anti-perilipin antibodies. Region of Interest (ROI) is shown in higher magnification. (H) Margin of the transplant from each mice was imaged at 20 × magnification and perilipin positive and negative cells were counted using ImageJ cell counter plugin and shown as percentage positive cells (n = 6). (I–K) ASCs infected with either Mock or DIRAS3 overexpressing lentiviruses were subjected to adipogenesis and mRNA expression of adipocyte marker genes normalized to actin was analyzed using q-RT-PCR at the indicated time points (I). Perilipin protein analysis (J) and Oil-Red-O staining (K) were done at day 9 post-induction of adipogenesis. All error bars represent the means ± SEM. p values * = p < 0.05, ** = p < 0.001 and *** = p < .0001. The significance of difference between means was assessed by analysis of variance (ANOVA) (A, C and I) and Student's t test (H).