Fig. 5

S1g-2 kills tamoxifen-resistant cells through Hsp70/Bim/ERα36 mediated signaling pathway. A Western blot of Hsp70 complexes using an H222 antibody that can recognize both ERα66 and ERα36, as well as Bim antibody in MCF-7/R1 cells upon treatment with 5 μM S1g-2 treatment for 0, 6, 12 or 24 h. B, C Western blot analysis of the levels of ERα66 and ERα36 using respectively specific monoclonal antibody, as well as PARP cleavage in MCF-7/R1 cells upon treatment with 5 μM S1g-2 treatment for 0, 6, 12 or 24 h. D Western blot analysis of the levels of ERα36 and ERα66 using respectively specific monoclonal antibody in MCF-7/R1, MCF-7/R2, MCF-7/TAM-R and T47D/TAM-R cells upon treatment with S1g-2 (5 μM) for 12 h, using β-actin as a loading control. An equivalent of DMSO was added to the compound untreated group as vehicle control. Bottom: the percentage inhibition of ERα36 and ERα66 upon S1g-2 treatment. The data are expressed as mean ± SD (n = 3 biologically independent experiments). *P < 0.05, **P < 0.01 (one-way ANOVA test); n.s. indicates no significance. E Western blot analysis of the levels of EGFR in MCF-7/TAM-R and T47D/TAM-R cells upon treatment with vehicle control, S1g-2 (5 μM), VER-155008 (5 μM) and PU-H71 (1 μM) for 12 h, using β-actin as a loading control. Bottom: Relative mRNA level of EGFR determined by real-time qPCR. β-actin gene was used as an endogenous control for normalization. The data are expressed as mean ± SD (n = 3 biologically independent experiments). *P < 0.05, **P < 0.01 (one-way ANOVA test)