Figure 3

Estrogen promoted the expression of Gli1 through noncanonical Shh signaling in MCF-7 cell lines. (A & C) Western blotting was used to detect (A) Gli1 and Shh expression in MCF-7 or (C) MDA-MB-231 cells incubated with 10 nM estrogen (E2) with or without 1 μM 4-hydroxy tamoxifen (4OHT) for 4 days. β-Actin was used as a loading control. In (B) MCF-7 or (D) MDA-MB-231 cells, mRNA expression levels of Gli1 and Shh were measured using qRT-PCR, and expression was normalized to that of GAPDH. (E) Western blotting was used to detect Gli1 protein expression in E2 and/or cyclopamine-treated MCF7 cells. (F) RT-PCR was used to detect Gli1 mRNA expression in E2 and/or cyclopamine-treated MCF7 cells. (G) Schematic presentation of three regions relative to the Gli1 transcriptional start site used to design primers to test for ER-α occupied abundance. (H) QChIP was performed to assess ER-α occupancy in ETOH and E2-treated MCF7 cells. (I) IgG was used as negative control. “% of input” indicates the ratio of DNA fragment of each promoter region bound by ER-α to the total amount of input DNA fragment without ER-α antibody pull-down. All data correspond to the mean ± SD of three independent experiments. **, ## indicate significant differences from the control (p < 0.001).