Fig. 3.

 IFI202b overexpression induces Zfp423 mRNA levels, accelerates adipogenesis and increases lipid storage in 3T3-L1 adipocytes. (a) Adenoviral-mediated overexpression of IFI202b (Ad-Ifi202b) was performed 24 h before a differentiation cocktail was applied. An empty adenovirus (Ad-empty) was used as a control. Cells were harvested at the indicated time points for quantitative real-time PCR, western blot and triacylglycerol (TG) analysis. (b) Overexpression of c-Myc-tagged IFI202b in 3T3-L1 cell lysates at the indicated time points. GAPDH was used as a loading control. (c–e) Gene expression levels of (c) Zfp423 and (d) Pparg at indicated time points and of (e) adipocyte-specific genes (at 72 h) in control and IFI202b-overexpressing 3T3-L1 pre-adipocytes (non-target siRNA, n = 5; si-Ifi202b, n = 6). (f) Triacylglycerol content in virus-infected 3T3-L1 cells was measured at day 6 of differentiation (n = 12). (g) Frequency of lipid droplets at the indicated sizes in differentiated 3T3-L1 adipocytes after adenoviral-infection with Ad-empty or Ad-Ifi202b was detected by automated imaging acquisition (16 images per well, n = 5). (c–g) White bars/circles, Ad-empty; black bars/circles, Ad-Ifi202b. (h, i) IFI202b expression was suppressed by transfecting confluent 3T3-L1 cells using specific siRNA. One day after electroporation, differentiation was induced and cells were harvested (h) 2 days later for quantitative real-time PCR analysis of the indicated adipocyte marker genes and (i) 5 days later for detection of triacylglycerol levels (non-target siRNA, n = 5; si-Ifi202b, n = 6). (h–i) White bars, non-target siRNA; black bars, Ifi202b siRNA (si-Ifi202b). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001, Student’s t test or two-way ANOVA with Sidak’s multiple comparisons test