Fig. 4.

The mechanism of E2 regulated NOTCH1. a, Western blot analysis of the indicated proteins in LNCaP-abl or PC3 cells with ERα knockdown (n = 3). b, Western blot analysis of the indicated proteins in LNCaP-abl or PC3 with or without ERα knockdown after treatment with either DMSO or 10 nM E2 for 72 h (n = 3). c, Diagram of the EZH2 promoter regions analyzed for EREs in the ChIP assays. d-f, Anti-ERα antibodies were used for ChIP assays regarding the EREs (ERE1-ERE4) of the NOTCH1 promoter in LNCaP-abl (d), LNCaP-abl with ERα knockdown (e), and LNCaP-abl treated with DMSO or E2 for 30 min before being harvested (f). qRT-PCR amplification was performed using a series of primers targeting EREs. The data are presented as the mean ± SD (n = 3). *, P < 0.05 vs. IgG (d), siNC (e), or DMSO (f). g, Anti-EZH2 antibodies were used for ChIP assays regarding the ERE2 and ERE3 of the NOTCH1 promoter in LNCaP-abl. The data are presented as the mean ± SD (n = 3). *, P < 0.05 vs. IgG. h, Co-IP analysis of the interaction between ERα and EZH2 in LNCaP-abl or PC3 cells following treatment with either DMSO or 10 nM E2 for 72 h (n = 3). Abbreviation: abl: LNCaP-abl. i and j, ChIP-re-ChIP qPCR analysis of ERE3 of NOTCH1 promoter in LNCaP-abl (i) and ChIP qPCR analysis of LNCaP-abl with ERα or EZH2 knockdown (j). The data are plotted as the mean ± SD (n = 3). *P < 0.05 vs. IgG (i) and siNC (j). k and l, Western blot analysis of the indicated proteins in LNCaP-abl with ERα or/and EZH2 knockdown (n = 3)