Fig. 2: RNF208 expression was transcriptionally activated by ERα in luminal breast cancer cells. | Nature Communications

Fig. 2: RNF208 expression was transcriptionally activated by ERα in luminal breast cancer cells.

From: RNF208, an estrogen-inducible E3 ligase, targets soluble Vimentin to suppress metastasis in triple-negative breast cancers

Fig. 2

a Comparison of RNF208 expression between ERα-positive and -negative breast cancer samples in published microarray datasets (GSE2034 and GSE5460). b RT-PCR (top) and immunoblot (bottom) analysis of ERα target genes (FOXOM1, GREB1, ESR1) and RNF208 expression in EtOH- or E2-treated ERα-positive breast cancer cells. 10 nM or 20 nM of E2 was treated in MCF-7 or T47D cell, respectively and E2 was treated for 24 h for RT-PCR and 48 h for immunoblot analysis. GAPDH and β-actin were used as internal controls. c Real-time quantitative RT-PCR (qRT-PCR) of RNF208 expression in ESR1-knockdown T47D cells upon E2 treatment. d Illustration of luciferase reporters including ERα-binding sites in the RNF208 promoter sequences. e T47D cells were transfected with various deletion constructs of the RNF208 promoter and then treated with or without E2 for 24 h. After E2 treatment, cells were assayed for luciferase activity. f ChIP analysis showing the recruitment of ERα to the human RNF208 promoter in E2-treated T47D cells. Precipitation was conducted with antinormal IgG or anti-ERα antibodies. g T47D cells were transfected with pGL3 control, RNF208 promoter, or its mutant (mtR4) plasmids containing mutated estrogen-responsive element site (CACC sequence replaced by GAAA) and then subjected to luciferase assays. h RT-PCR and immunoblot analysis showing RNF208 expression upon E2 treatment for 24 h (mRNA) or 48 h (protein), respectively, in control or ERE knockout MCF-7 cells. i Immunoblot analysis of ERα with 5-aza-dC treatment in TNBC cells. Hs578T and MDA-MB-231 cells were treated with 10 μM 5-aza-dC for 96 h and β-actin was used for normalization. j TNBC cells were transiently transfected with 20 nM of control siRNA or ESR1 siRNA and then treated with 10 μM 5-aza-dC for 96 h. Cell lysates were immunoblotted with the indicated antibodies. All P values were calculated by unpaired two-tailed Student’s t tests (e, g). These data represent the mean ± SD of three independent experiments. Source data for (c, e, g, h) are available in Source Data file. Unprocessed original scans of blots and gels in (b, f, h−j) are shown in Supplementary Fig. 13.

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