FIGURE 5.

I-BET151 reduces Brd4 occupancy of the Gli1 locus. A, knockdown of Brd4 inhibits HH target gene expression. 50 nm Brd4 siRNA or scramble siRNA was transfected into Sufu^−/− MEFs for 72 h. RNA was subsequently extracted for quantitative PCR to determine the expression of the indicated genes. B, schematic of the mouse Gli1 locus from −10,000 bp to +500 bp, relative to the transcription start sites (TSS) shown. Brd4 occupancy was analyzed by ChIP-quantitative PCR in Sufu^−/− MEFs treated with 0.5 μm I-BET151 and then normalized to a control ChIP performed using rabbit IgG. C, a plasmid expressing human GLI1 from a CMV promoter was transfected into Light2 cells and then treated with 0.1 or 0.5 μm I-BET151 48 h later. After 24 h of treatment, firefly luciferase activity was measured and normalized to Renilla luciferase activity, followed by isolation of RNA to measure the expression of the indicated HH target genes. Error bars represent the S.E. of three independent experiments. p values ≤ 0.05 are considered statistically significant and indicated by an asterisk. NS, not significant.