Skip to main content
. 2020 Feb 10;10(8):3382–3396. doi: 10.7150/thno.40144

Figure 4.

Figure 4

ERRγ interacts with p65 to regulate ABCB1 transcription. (A) Schematic representation of ERREs in the promoter region of ABCB1 with changes of nucleotides in ERRE1 and ERRE2 shown as indicated; (B) ChIP-PCR assay showing ERRγ binding to ERRE1 and ERRE2 in ABCB1 promoter. The input (5%), binding between ERRγ and the promoter of ABCB1 at the potential binding site ERRE1/2, was amplified by qPCR (right) and confirmed by 2% agarose gel electrophoresis (left); (C) Schematic representation of mutated ERRE positions in pGL-ABCB1 vector; (D) Reporter gene assay performed in HepG2 cells 24 h post transfection with pGL-ABCB1-WT or pGL-ABCB1-Mut1/2/3 by dual-luciferase analysis; (E) Examination of ERRγ interaction with different transcription factors in HepG2/ADR and MCF-7/ADR cells following immunoprecipitation with ERRγ or control antibody and analyzed by Western blot analysis; (F) Interaction between ERRγ and p65 in HepG2 and HepG2/ADR cells monitored by immunoprecipitation using anti-ERRγ antibody; After ERRγ was immunoprecipitated, the binding between ERRγ and p65 was examined by Western blot analysis. An equal amount of ERRγ was loaded for normalization according to a pre-Western blot; (G) Expression and localization of p65 (green) and ERRγ (red) in HepG2 and HepG2/ADR cells visualized by confocal imaging; (H) Interaction between ERRγ and p65 in HepG2/ADR cells treated with or without BAY 11-7082 for 12 h and then analyzed by immunoprecipitation using an antibody against ERRγ; (I) Dual-luciferase reporter gene assay performed in HepG2 cells transfected with pGL-ABCB1-WT or pGL-ABCB1-Mut1/2, with or without pcDNA/ERRγ, for 12 h and then further treated with or without BAY 11-7082 for 12 h; (J) HepG2/ADR cells were treated with si-RNA or si-ERRγ combined with or without BAY 11-7082 for 12 h and then further treated with 5 μM Dox for 48 h; (K) Model for ERRγ/p65-promoted transcription of ABCB1 in chemoresistant cancer cells. Data were presented as means ± SD from three independent experiments. **p< 0.01. NS, no significant.