Fig. 4: RNF181 facilitates ERα signaling in breast cancer cells.

a RNF181 consumption decreased ERα protein levels in MCF-7 cells. MCF-7 cells were transfected with siControl or siRNF181. After 48 h, cells were harvested for western blot analysis. RNF181 and ERα protein levels were determined by Western blot. Actin was used as internal control. b RNF181 consumption decreased ERα target gene expression in MCF-7 cells. MCF-7 cells were transfected with siControl or siRNF181. After 48 h, total RNA was extracted for gene expression analysis. *P < 0.05; **P < 0.01; ***P < 0.001 for target gene expression comparison. c RNF181 depletion decreases ERα protein levels in both vehicle and E2-treated conditions in MCF-7 cells. MCF-7 cells were transfected with siRNF181 or siControl. After 48 h, cells were treated with either ethanol or 10 nM estradiol for 6 h. RNF181 and ERα protein levels were determined by Western blot analysis. Actin was used as internal control. d RNF181 depletion decreases ERα protein levels in both vehicle and E2-treated conditions in T47D cells. T47D cells were transfected with siRNF181 or siControl. After 48 h, cells were treated with either ethanol or 10 nM estradiol for 6 h. RNF181 and ERα protein levels were determined by Western blot analysis. Actin was used as internal control. e RNF181 depletion decreases ERα target genes in both vehicle and E2-treated conditions in MCF-7 cells. MCF-7 cells were transfected with siRNF181 or siControl. After 48 h, cells were treated with either ethanol or 10 nM estradiol for 6 h. Total RNA was prepared and the expression of the endogenous ER alpha target genes, PS2, GREB1, and PDZK1 were determined by qPCR. Shown are the results from three experiments. *P < 0.05; **P < 0.01; ***P < 0.001 for target gene expression comparison. f RNF181 depletion decreases ERα target genes in both vehicle and E2-treated conditions in T47D cells. T47D cells were transfected with siRNF181 or siControl. After 48 h, cells were treated with either ethanol or 10 nM estradiol for 6 h. Total RNA was prepared and the expression of the endogenous ER alpha target genes, PS2, GREB1, and PDZK1 were determined by qPCR. Shown are the results from three experiments. *P < 0.05; **P < 0.01; ***P < 0.001 for target gene expression comparison. g RNF181 depletion affects ERE-luciferase activity in MCF-7 cells. MCF-7 cells were transfected with siRNF181 or siControl together with ERE luciferase reporter plasmid. Cells were treated with 10 nM estradiol or vehicle. Luciferase activity was measured 48 h after transfection. Shown are the results from three experiments. *P < 0.05; **P < 0.01; ***P < 0.001 for luciferase activity comparison. h RNF181 depletion affects ERE-luciferase activity in T47D cells. T47D cells were transfected with siRNF181 or siControl together with ERE luciferase reporter plasmid. Cells were treated with 10 nM estradiol or vehicle. Luciferase activity was measured 48 h after transfection. Shown are the results from three experiments. *P < 0.05; **P < 0.01; ***P < 0.001 for luciferase activity comparison. i RNF181 depletion decreases ERα target genes in both vehicle and tamoxifen-treated conditions in MCF-7 cells. MCF-7 cells were transfected with siRNF181 or siControl. After 48 h, cells were treated with either ethanol or 0.5 uM tamoxifen for 6 h. Total RNA was prepared and the expression of the endogenous ER alpha target genes, PS2, GREB1, and PDZK1 were determined by qPCR. Shown are the results from three experiments. *P < 0.05; **P < 0.01; ***P < 0.001 for target gene expression comparison. j RNF181 depletion sensitizes tamoxifen inhibition effect in MCF-7 breast cancer cells. MCF-7 cells were transfected with siRNF181 or siControl. After 48 h, cells were plated into 96-well plate, while each well contained 4000 cells. The indicated tamoxifen concentrations were used for 48 h. The numbers of the cells were determined via CCK8 kit for the cellar metabolic activity. Experiments were done in triplicates. *P < 0.05; **P < 0.01; ***P < 0.001 for cell growth comparison.