Figure 8.

siRNA directed against c-Myc blocks the glucose-mediated recruitment of ChREBP to the Pklr gene. INS-1-derived 832/13 cells were transfected with siRNA specific for c-Myc or a control siRNA and were cultured for 48 h. Cells were then treated for 16 h with either 2 or 20 mm glucose as in Fig. 2. A, Nuclear extracts were subjected to immunoblotting with an antibody directed against c-Myc or β-actin. The result is representative of two independent experiments. B, Total RNA was isolated and subjected to RT-PCR using primers specific for Pklr and β-actin. C, Alternatively, cells were fixed with formaldehyde, chromatin was isolated and sheared, and protein-DNA complexes were immunoprecipitated with antibodies directed against ChREBP; the DNA was purified and amplified by PCR using the indicated primer pairs. The results from panels B and C are expressed as the mean of four experiments, ± se. #, P < 0.05 for 2 mm glucose vs. 20 mm glucose; *, P < 0.05 for 20 mm glucose siRNA control vs. 20 mm glucose and siRNA specific for c-Myc; §, P < 0.05 for 2 mm glucose c-Myc siRNA vs. 20 mm glucose c-Myc siRNA; ns, not significant. Si Control, Small interfering control; si-c Myc, small interfering Myc.