Figure 2.

Rb depletion compromises global AR repression activity

(A) Immunoblotting for Rb in C4-2-tet-shRB cells treated with doxycycline (0.05 μg/mL) at 0, 3, or 30 d (days). (B) The flow cytometry cell cycle analysis for C4-2-tet-shRB cells treated with or without doxycycline and with or without 10 nM DHT for 24 h. (C) RNA-seq analyses were done in C4-2-tet-shRB cells treated with doxycycline (0.05 μg/mL at 0, 3, or 30 days) and stimulated with or without DHT (10 nM, 24 h). Heatmap view for DHT-repressed genes was shown. (D) Box plots for the change of expression (Log₂(fold-change)) for E2F target genes or Myc target genes in these samples are shown. (E) Heatmap view for E2Fs (upper panel) and a panel of E2F target genes (lower panel) is shown. (F) Heatmap view for MYC and its coregulators (upper panel) and a panel of Myc target genes (lower panel) is shown. (G) Immunoblotting for Rb, AR, and FOXA1 in control C4-2 cells versus selected clones of C4-2 with RB1 knockout (KO1–4) using CRISPR-Cas9 approach is shown. (H) The proliferation assay for control C4-2 cells and two RB-KO lines treated with 0–20 μM of enzalutamide for 6 d is shown. (I) The flow cytometry cell cycle analysis for the control C4-2 and two RB-KO clones is shown. (J) Quantitative real-time PCR for several E2F-regulated genes in control lines versus RB-KO lines is shown. Data in bar graphs represent the mean ± SD. n.s., not significant. ∗p < 0.05.