Fig. 4

BRMS1 inhibits TGF-β1-induced HIF-1α expression. a The cells were transfected with BRMS1 or empty vector (control) for 48 h and then stimulated with TGF-β1 (5 ng/ml) for 6 h. HIF-1α mRNA expression was analyzed by quantitative RT–PCR. Relative HIF-1α mRNA levels normalized to the expression of the housekeeping gene, GAPDH (*P < 0.05 and **P < 0.01 vs control, ^#P < 0.05 and ^##P < 0.01 vs control with TGF-β1). b The MDA-MB-231 cells were co-transfected with the empty vector (control), BRMS1 and HIF-1α reporter plasmids for 48 h, followed by stimulation with TGF-β1 (5 ng/ml) for 24 h. Luciferase activity (**P < 0.01 vs control, ^##P < 0.01 vs control with TGF-β1). c The MDA-MB-231 cells were transfected with BRMS1 or empty vector (control) for 48 h and then stimulated with TGF-β1 (5 ng/ml) for 6 h. Immunoblotting. d The MDA-MB-231 cells were sequentially transfected with indicated vectors for 48 h and then stimulated with TGF-β1 (5 ng/ml) for 6 h. The cells were fixed and stained to assess expression of HIF-1α. e The MDA-MB-231 cells were transfected with indicated vectors before TGF-β1 (5 ng/ml) treatment. The ChIP assay was performed as described in Materials and Methods. Representative results were presented from at least three independent experiments with similar results