MIBE inhibits GPER target genes and proliferation induced by E2, G-1 and OHT. (a) The expression of c-fos, CTGF, Cyr61 and EGR1 induced in SkBr3 cells by 1 h treatment with 100 nM E2, 1 microM G-1 and 5 microM OHT is inhibited in presence of 10 microM MIBE, as evaluated by real-time PCR. Results obtained from experiments performed in triplicate were normalized for 18S expression and shown as fold change of RNA expression compared to cells treated with vehicle. Each data point represents the mean ± SD of three independent experiments performed in triplicate. (b) The up-regulation of c-fos and CTGF protein levels induced in SkBr3 cells by 2 h treatment with 100 nM E2, 1 microM G-1 and 5 microM OHT were abolished in presence of 10 microM MIBE. Data shown are representative of three independent experiments. beta-actin serves as a loading control. (c) Densitometric analysis of c-fos and CTGF protein expressions normalized to beta-actin. (•), (◦) indicate P < 0.05 for cells receiving vehicle versus treatments. (d) The proliferation of SkBr3 cells upon treatment with 100 nM E2, 100 nM G-1 and 100 nM OHT was inhibited by 1 microM MIBE, as indicated. Cells were treated for five days with the indicated treatments and counted on Day 6. Proliferation of cells receiving vehicle was set as 100% upon which cell growth induced by treatments was calculated. Each data point is the average ± SD of three independent experiments performed in triplicate. (•), (◦), (▪), indicate P < 0.05 for cells receiving vehicle (-) versus treatments.