Fig. 7: ERα transcriptionally regulates PSMD14 expression, which forms a forward regulation loop between ERα signaling and PSMD14.

A The ChIP-seq analysis of ER binding to the PSMD14 promoter region utilized data from GEO with accession numbers GSE128208. B ChIP assay showed that ERα could bind to the promoter region of PSMD14. MCF-7 cells were fixed for 30 min. The Rabbit IgG was used as the negative control. The primer sequence was shown in method section. The enriched DNA fragments were subject to PCR reaction and DNA gel electrophoresis. qRT-PCR analysis showing mRNA levels of ERα after ERα depletion. MCF-7 (C) and T47D (D) cells transfected with 50 nM siControl or two independent siERα. After 48 h, total RNA was extracted for gene expression analysis. E, F ERα depletion in MCF-7 and T47D cells inhibited PSMD14 mRNA. MCF-7 and T47D cells were transfected with 50 nM siControl or siERα. After 48 h, total RNA was extracted for gene expression analysis. The relative TFF1 (positive control for ERα depletion) and PSMD14 mRNA levels were assessed by qRT-PCR. Each group was tested in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 for comparisons of target gene expression. G, H ERα depletion decreases PSMD14 protein level. MCF-7 and T47D cells were transfected 50 nM siControl or siERα. Cell lysates were immunoblotted with the indicated antibodies. β-Actin was used as internal control. I, J ChIP assay showed that ERα depletion decreases ERα recruitment to PSMD14 promoter. ERα was depleted in MCF-7 and T47D cells, and ChIP-qPCR assays showed that ERα reduced binding to PSMD14 gene. K, L E2-treated in MCF-7 and T47D cells increased PSMD14 mRNA. MCF-7 and T47D cells were treated with 10 μM E2 for the indicated times. Total RNA was extracted for gene expression analysis. The relative TFF1 (positive control for E2-treated) and PSMD14 mRNA levels were assessed by qRT-PCR. Each group was tested in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 for comparisons of target gene expression. M, N E2-treated in MCF-7 and T47D cells increased PSMD14 protein level. MCF-7 and T47D cells were treated with 10 μM E2 for the indicated times. Cell lysates were immunoblotted with the indicated antibodies. β-Actin was used as internal control. O, P ChIP assay showed that E2-treated increases ERα recruitment to PSMD14 promoter. MCF-7 and T47D cells were stimulated with E2 for 30 min and ChIP-qPCR assay showed increased binding of ERα to the PSMD14 gene. Data are shown as mean ± SD, N = 3. Two-tailed t test. *P < 0.05; **P < 0.01; ***P < 0.001.