Figure 3
From: Foxm1 controls a pro-stemness microRNA network in neural stem cells
Foxm1 promoter occupancy by Gli1 and Gli2. (A) Schematic of the Foxm1 promoter showing locations of the 8 putative Gli-responsive elements (s1–s8). (B,C) qPCR-ChIP assay of endogenous Gli1 and Gli2 occupancy of the Foxm1 promoter region in NSCs and Diff-NSCs. Immunoprecipitation with IgG was performed as control. Anti-acetyl-H3 antibodies was used to detect Foxm1 transcriptional activation. Eluted DNA was qPCR-amplified using primers encompassing putative Gli binding sites [s1–s5 (B) and s6–s8 (C)]. Results are expressed as fold induction values relative to ChIP input controls. B-actin was utilized as unrelated chromatin control and is presented in Supplementary Figure 5 A. Bars represent means (SD) of three independent experiments. P values vs. Diff-NSCs (Mann-Whitney U test): (B) *P < 0.05: 0.04797 (s1-5, Gli2), 0.03271 (s1-5, AcH3); NS (not significant): 0.2514 (s1-5, Gli1). (C) *P < 0.05: 0.0490 (s6-8, AcH3); **P < 0.01: 0.001374 (s6-8, Gli1); NS: 0.296763205 (s6-8, Gli2). (D) Luciferase activity induced in the Foxm1 promoter region in NSCs by Gli1, Gli2, and Mock (negative control, PCDNA). Results are normalized to pRL-CMV-Renilla luciferase (R-Luciferase). Bars represent means (SD) of at least three independent experiments, each performed in triplicate. P values vs. control cells (One-way ANOVA test): *P < 0.05: 0.02 (Gli wt-Gli1); **P < 0.01: 0.005 (Gli wt-Gli2), NS: Not significant 0.072 (Mut Gli s1-5-Gli1); 0.066(Mut Gli s1-5-Gli2); 0.083 (Mut Gli s6-8-Gli1); 0.077(Mut Gli s6-8-Gli2).
