Fig. 3

SMURF1 depletion or inhibition in breast cancer cells decreases ER alpha signaling activity. a SMURF1 depletion effect by two different siRNA oligos. MCF-7 cells are transfected with two independent SMURF1 siRNAs or siControl. After 48 h, SMURF1 mRNA levels are determined by QPCR. 36B4 was used as internal control. *P < 0.05; ** P < 0.01; ***P < 0.001 for SMURF1 mRNA level comparison. b and c SMURF1 depletion effect on ERα protein level by two different siRNA oligos. MCF-7 or T47D cells were transfected with siSMURF1 or siControl. After 48 h, cells were treated with either ethanol or 10 nM estradiol for 6 h. SMURF1 and ERα protein levels were determined by Western blot analysis. Actin was used as internal control. d and e SMURF1 depletion affects ERE-luciferase activity in MCF7 and T47D cells. MCF7 or T47D cells were transfected with siSMURF1 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. f SMURF1 depletion decreases ERα target genes using two different siRNA oligos. MCF-7 cells were transfected with siSMURF1 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 ERα 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 and h SMURF1 inhibitor A01 decreases ER alpha protein in breast cancer cells. MCF-7 and T47D cells were both treated with 10 nM E2 or vehicle for 24 h and then continue with 10 uM A01 or vehicle 12 h,ER alpha and SMURF1 levels were determined by western blot analysis. β-actin was used as internal control. i and j SMURF1 inhibitor A1 decrease ERE-luciferase activity in MCF7 and T47D cells. MCF7 or T47D cells were transfected with ERE luciferase reporter plasmid. Cells were both treated with 10 nM E2 or vehicle for 24 h and then continue with 10 uM A01 or vehicle for 12 h. Luciferase activity was measured. Shown are the results from three experiments. *P < 0.05; ** P < 0.01; ***P < 0.001 for luciferase activity comparison