Fig. 3: Bufalin induces ERα degradation and suppresses its transcriptional activity.

a MCF-7 and T47D cells were treated with a series of concentrations of Bufalin for 48 h, and the expression of ERα was measured by western blot. b MCF-7 and T47D cells were treated with 50 nM Bufalin for different periods of time, and the expression of ERα was measured by western blot. c MCF-7 cells were treated with Bufalin, and then subjected to cycloheximide (CHX) (10 μg/ml) chase at the indicated time, the expression of ERα was measured by western blot. Representative data are shown from n = 3 independent experiments with consistent results. d MCF-7 and T47D cells were treated with Bufalin for 48 h with or without MG-132. The expression of ERα was measured by western blot. e MCF-7 and T47D cells were treated with Bufalin for 48 h in the presence or absence of MLN4924. The expression of ERα was measured by western blot. f MCF-7 and T47D cells were treated with Bufalin in the presence or absence of hydroxychloroquine (HCQ). The expression of ERα and LC3 was measured by western blot, the samples derive from the same experiment, and the gels/blots were processed in parallel. g 293 T cells were transfected with ERα plasmid and HA-Ub plasmid, and then subjected to Bufalin for 48 h, followed by treatment with MG-132 (10 μM) for 10 hours before harvest. Then the cells lysates were subjected immunoprecipitation with anti-ERα antibodies and blotted with anti-HA antibodies. h 293 T cells were transfected with Flag-ERα plasmid, and then subjected to Bufalin for 48 h, followed by treatment with MG-132 (10 μM) for 10 hours before harvest. Then the cells lysates were subjected immunoprecipitation with anti-Flag antibodies and blotted with anti-Ub antibodies. i ERE-luciferase assay after Bufalin treatment, the data are presented as mean ± SD, n = 3 independent experiments. Two-way ANOVA was used for statistical analysis, P < 0.05 was considered to be statistically significant. j MCF-7 cells were treated with a series of concentrations of Bufalin for 48 h in the presence or absence of E2, the mRNA levels of AGR2, CCND1, GREB1, NRIP1, PGR, and SIAH2 were analyzed by real-time PCR, the data are presented as mean ± SD, n = 3 independent experiments. Two-way ANOVA was used for statistical analysis, P < 0.05 was considered to be statistically significant.