Fig. 4.

DIRAS3 knock-down mediated enhancement of adipogenesis is orchestrated by up-regulation of C/EBP-β and PPAR-γ2. (A) Relative expression of CEBP-β, PPAR-γ2 and CEBP-α was analyzed normalized to actin at different time points during adipogenesis by RT-PCR. (B) CEBP-β protein level during 72 h after adipogenesis induction. For optimal separation of bands, C/EBP-β Full-LAP and C/EBP-β LAP were analyzed using a 8% PAGE (upper panel) and C/EBP-β LIP using a 12,5% PAGE (lower panel). (C and D) Phosphorylation of ERK1/2 (T282/Y204) (C) and Foxo-1(S256) (D) was monitored by Western blotting during 72 h of adipogenesis. (E) Localization of Foxo-1 within the cells upon DIRAS3 KD was visualized by IF-CLSM. Foxo-1 (green), TO-PRO3 for nuclear staining (red), Merge (white, obtained by using a co-localization mask). Co-localization mask was obtained by plotting gray scale pixels in x–y axis scatter plot, where x-axis represents green channel (Foxo1) and y-axis represents red channel (TO-PRO3). Double positive pixels are represented in co-localization mask as white. Laser Sharp 2000 software from Zeiss was employed for the image analyses. 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).