FIGURE 5.
Changes in FoxO1 level modify mouse Ins2 gene expression. A, Western blot showing protein extracted from MIN6 cells transfected with the indicated siRNA. MIN6 cells were transfected with Smart Pool or scrambled siRNA (20 nm) for 48 h before protein analysis. B, real-time qRT-PCR analysis of the indicated genes after siRNA-mediated silencing of FoxO1 studied at 3 and 30 mm glucose concentrations. MIN6 cells were transfected with indicated siRNA as mentioned above. Subsequently, cells were cultured in 3 mm glucose for overnight prior to incubation in 3 or 30 mm glucose for 6–8 h and RNA was extracted for real-time RT-PCR analysis. C, MIN6 cells overexpressing a constitutively active (nuclear) FoxO1-CA-EGFP were analyzed by real-time RT-PCR of the indicated genes, as in B. Cells were transduced with FoxO1-CA virus (20–25 MOI) for 12 h and subsequently cultured overnight (16 h) in 3 mm glucose prior to incubation in 3 or 30 mm glucose for 6–8 h and RNA was extracted for analysis (see “Experimental Procedures”). Bars (B, C) show mean ± S.E., n≥ 9 from 4–5 independent experiments of the ddCT values (compared with control samples in 30 mm glucose for each gene).