Fig. 6
LEM4 induces ERα transactivation activity. a Immunoblot analysis of the phosphorylation of ER, Cyclin D1, and c-Myc in MCF-LEM4 and MCF7-TAMR cells. b Immunoblot analysis of the phosphorylation of ER, Cyclin D1, and c-Myc in LEM4-depleted MCF7-LEM4 and LEM4 knocked-down MCF7-TAMR cells. c Luciferase assay. ER+ MCF7 and ERα-negative MDA-MB-231 cells were transfected with ERE-Luc and other indicated plasmids. Following incubation for 48 h, luciferase activity was measured and normalized to Renilla. Results are the mean ± s.e.m. of three independent experiments performed in triplicate. d ERα ChIP assay of known ER-binding sites in ERα target genes was performed in MCF7-LEM4 cells incubated in estrogen-depleted medium (5% charcoal-stripped serum in phenol red-free DMEM) for 72 h before treatment with vehicle, 10 nmol L−1 E2 for 45 min. e Time course ChIP study of the endogenous ERα with the estrogen response elements in the promoter region of the ERα target genes. MCF7, MCF7-LEM4, and LEM4-depleted MCF7-LEM4 cells incubated in estrogen-depleted medium (5% charcoal-stripped serum in phenol red-free DMEM) for 72 h before treatment with vehicle, 10 nmol L−1 E2. ChIP analysis was conducted by using anti-ERα antibody. *P < 0.05, **P < 0.01, ***P < 0.001. Tukey’s multiple comparisons test for c. Student’s t-test for d
