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. 2023 Feb 2;51(6):2655–2670. doi: 10.1093/nar/gkad043

Figure 5.

Figure 5.

Overlaps of SMAD3 peaks and AR peaks in the ChIP-seq analysis. (A) Co-immunoprecipitation (Co-IP) of AR with SMAD3 in Rv1 or C4-2 cells. SMAD3 was immunoprecipitated from cells and analyzed by western blotting for co-precipitation of AR. Trueblot secondary antibodies were used in the western blots. (B) The C-terminal MH2 domain of SMAD3 interacted with AR. Myc-AR was co-expressed with Flag-tagged SMAD3 fragments (WT, Δ C mutant lacking the C-terminal MH2 domain, or Δ N mutant lacking the N-terminal MH1 domain) in 293T cells. Flag IP was performed and analyzed by western blotting with Flag or myc antibodies. (C) N-TAD domain of AR interacted with SMAD3. Myc-SMAD3 was co-expressed with the Flag-tagged AR fragments (N-terminal transactivation domain, N-TAD; DNA binding domain, DBD; Ligand-binding domain, LBD) in 293T cells, and analyzed as described in (B). (D) Heatmap showing two replicates of AR ChIP-seq peaks relative to IgG negative control antibody. (E) AR ChIP-seq peak annotation. (F) The Homer motif analysis showing the significant enrichment of ARE or AR half-site motifs on AR peaks. (G) Venn diagram showing the overlap of AR peaks and SMAD3 peaks in the ChIP-seq analysis. Cut&Run ChIP-seq studies were performed on Rv1 cells using AR or SMAD3 antibodies. Peak calling identified 12745 AR peaks and 11779 SMAD3 peaks. The overlapping of AR and SMAD3 peaks was determined by the ChIPpeakAnno package in R. (H) Heatmap showing the AR enriched peaks, common peaks and SMAD3 enriched peaks. (I) GO analysis of the genes associated with the common peaks between AR and SMAD3. (J) Distribution of AR (blue) and SMAD3 (green) ChIP-seq peak signal near the common peak center. (K) Example signal track image showing the SMAD3 peaks and AR peaks at the KLK3 enhancer or KLK2 promoter, which is indicated with an arrow. (L and M) ChIP-PCR showing the enrichment of AR and SMAD3 at KLK3 enhancer (L) or KLK2 promoter (M) of C4-2 cells after androgen treatment. ChIP assays were performed with control, AR or SMAD3 antibodies. Precipitated chromatin was analyzed by qPCR for KLK3 enhancer or KLK2 promoter. % of input was calculated and presented as mean ± SD (n = 3), and ANOVA was used for statistical analysis (ns, not significant; ***P < 0.001).