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. 2019 Apr 10;20(7):1772. doi: 10.3390/ijms20071772

Figure 3.

Figure 3

RNF146 expression induced by rhododendrin is mediated by ERβ activation. (A) Quantification of relative RNF146 mRNA expression levels (normalized to those of GAPDH) in SH-SY5Y cells treated with rhododendrin (10 µM, 37 h) in the presence or absence of the ER inhibitor tamoxifen (1 µM, 8 h) determined by RT-qPCR (n = 3). (B) Representative Western blot showing that rhododendrin (10 µM, 37 h)-mediated induction of RNF146 expression is blocked by tamoxifen treatment (1 µM, 8 h). (C) Quantification of relative RNF146 expression levels (normalized to those of β-actin) in the experimental groups in panel B (n = 3). (D) Quantification of relative RNF146 expression levels (normalized to those of GAPDH) in SH-SY5Y cells treated with rhododendrin (10 µM, 37 h) determined by RT-qPCR. ERβ expression was knocked down by CRISPR-cas9-mediated deletion of ERβ (85 h). sgRNA to EGFP was used as CRISPR-cas9 transfection control (n = 3). (E) Representative Western blot showing that rhododendrin (10 µM, 37 h)-mediated induction of RNF146 expression is abolished by CRISPR-cas9 mediated deletion of ERβ (85 h). (F) Quantification of relative RNF146 and ERβ expression levels (normalized to those of β-actin) in the experimental groups in panel E (n = 3). (G) Representative immunofluorescence images showing nuclear translocation of ERβ in response to rhododendrin treatment (37 h) in SH-SY5Y cells. (H) Relative distribution of ERβ in the nucleus as normalized to ERβ in the total cell area (n = 30 cells from three independent experiments). (I) Chromatin anti-ERβ immunoprecipitation (ChIP) of a putative ER responsive element (ERβ motif) within the RNF146 promoter region determined by PCR using specific primers. Non-ER responsive elements within RNF146 promoter (Control motif) and the β-actin region were used as negative controls. Immunoprecipitation using either anti-histone antibodies or rabbit IgG was included as a ChIP experimental control. Note the increase of ERβ occupancy of the RNF146 promoter ERβ motif induced by rhododendrin treatment (10 µM, 37 h). Similar results were obtained from two independent experiments. Quantified data are expressed as mean ± SEM. Statistical significance was determined by unpaired two-tailed Student’s t-test (H) or ANOVA test with Tukey’s post-hoc analysis (A,C,D,F), ** p < 0.01, and *** p < 0.001.