Metformin (Met) inhibits E2-inducible ERE luciferase activity and expression of ERα target genes in MCF-7 and TR MCF-7 cells. (A) Transfected cells were treated with Met (25 mM) and 4-OHT (10−6 M) in the presence of E2 (100 nM) and ERE luciferase assay was performed. (B) MCF-7 (left) and TR MCF-7 (right) cells were treated with Met and 4-OHT in the presence of E2 for 48 h in estrogen-depleted RPMI-1640 medium containing 3% charcoal stripped FBS. The protein levels of c-Myc, cyclin D1, PR, pS2, phospho(p)-AMPKα (Thr172) and total AMPKα were examined by western blotting. β-actin was evaluated as a loading control. Protein expression levels normalized to β-actin are presented. Data represent the mean ± SD of three independent experiments. P-value was calculated compared to untreated Ctrl or E2-treated cells; *P<0.05 and **P<0.01. (C) RT-qPCR was conducted to assess the mRNA levels of ERα target genes, cyclin D1 and pS2. All data represent the mean ± SD of three independent experiments conducted in triplicates; *P<0.05 and **P<0.01.