Fig 2: Androgen-mediated repression of cell-cycle genes is dependent on RBL1 and RBL2. A, GSVA signature score heatmaps of gene expression signatures on RNA-seq data generated from LNCaP models treated with SPA (10 nmol/L R1881) for 48 hours. B, Heatmap of mean-centered log2 (FPKM) RNA-seq values for the androgen-upregulated AR Activity signature genes. LNCaP and LNCaP-DKO data are replotted from Fig. 1. C, Plot of GSEA enrichment scores of MSigDB Hallmark gene sets representing SPA-induced gene expression changes in LNCaPDKO and LNCaPDKO-RBL1/2 cells. D, Heatmap of mean-centered log2(FPKM) RNA-seq values for the cell-cycle progression (CCP) signature genes. LNCaP and LNCaP-DKO data are replotted from Fig. 1. E, Heatmap of mean-centered log2(FPKM) RNA-seq values for the top 10 SPA-repressed genes in the LNCaPDKO-RBL1/2 cells. LNCaP and LNCaP-DKO data are replotted from Fig. 1.

Fig 3: Cytosolic death effector domain-containing DNA-binding protein (DEDD) promotes retinoblastoma (Rb)/p107 proteasome degradation. a Western blots show Rb protein expression levels 6 h after abolishing synchronization by fetal bovine serum (FBS) in DEDD knockdown triple-negative breast cancer (TNBC) cell lines (HCC1806, HCC38, Rb intact). b Quantitative PCR showing both DEDD and Rb1 gene expression in DEDD-knockdown TNBC cell lines. c Western blots showing Rb protein expression in DEDD-knockdown HCC1806 cells under the MG-132 (proteasome inhibitor) treatment for indicated hours. d Western blots showing Rb protein expression levels in the 293FT cells treated with different dosages of 3×-FLAG-WT-DEDD- or 3×-FLAG-ΔNLS-DEDD-expressing plasmids with or without MG-132 treatment. e Western blots showing Rb protein expression levels in KTB-21 cells with or without the overexpression of cytosolic DEDD under the treatment of MG-132. f Immunoprecipitation assay showing the interaction between cytosolic DEDD and Rb protein after overexpressing 3×-FLAG-ΔNLS-DEDD in 293FT cells. g Immunoprecipitation assay showing Rb protein ubiquitination level after co-transfection of HA-ubiquitin and 3×-FLAG-ΔNLS-DEDD in 293FT cells. h Western blots showing Rb protein expression levels 6 h after abolishing synchronization by FBS in DEDD-knockdown TNBC cell lines (MDA-MB-468, Rb deficient). i Western blots showing Rb protein expression in DEDD-knockdown MDA-MB-468 cells under MG-132 treatment for indicated hours. All quantitative data were generated from a minimum of three replicates. P values were derived from one-way analysis of variance (ANOVA) with multiple comparison test. Error bars represent means ± s.e.m.

Fig 4: SPA repression of E2F target genes and prostate cancer proliferation depends on RBL1 and RBL2. A, E2F1 ChIP-seq volcano plot showing differentially bound E2F1 binding sites between EtOH- and SPA-treated LNCaPDKORB1−/−;TP53−/− cells. B, Overlap between RB1-bound sites in LNCaP and E2F1-bound sites in LNCaPDKO cells. These E2F1:RB1 bound sites were mapped to promoters of genes, which were overlapped with genes transcriptionally repressed by SPA. C, Graph of over-represented GO categories in overlapping RB1/E2F-bound and SPA-repressed genes. D, Growth assay results for knockout and parental cells treated with 10 μmol/L ENZ or SPA for 5 days. E–G, Cell-cycle profile changes induced by SPA on LNCaP (E), LNCaPDKO (F), or LNCaPDKO;RBL1/2 (G) cells using flow cytometry to measure EdU incorporation (S-phase) and FxCycle violet staining (DNA-content). H, R1881 dose–response assay comparing LNCaPDKO cells with LNCaPDKO;RBL1/2 cells with and without ectopic expression of RBL2. I, R1881 dose–response assay on VCAP and the VCAPRB1–14 RB1-knockout clonal cell line transfected with siRNA to RBL1/2 or a nontargeting control (NTC). *, P < 0.05, by one-way ANOVA with Dunnett multiple-comparison test. Data represent the mean ± SD.