Fig 2: Functional analysis of CRISPR/Cas9-modified PCa cells with different rs2853669 genotype. (A) Sanger sequencing of CRISPR/Cas9-modified and parental 22Rv1 cells. (B) Chromatin enrichment of E2F1 and MYC at the rs2853669 site measured by ChIP-qPCR. (C) Western blot showing the protein levels of E-cadherin and N-cadherin in CRISPR/Cas9-modified and parental 22Rv1 cell lines. GAPDH was used as a loading control. (D) Representative microscopy analysis of E-cadherin (green; upper panels) and N-cadherin (green; lower panels) expression in CRISPR/Cas9-modified and parental 22Rv1 cell lines. Nuclei were counterstained with DAPI (blue). Scale bars, 20 µm. (E, F) Cell proliferation analysis of the TT genotype, TC genotype, and CC genotype at rs2853669 in 22Rv1 cells under androgen stimulation (E) or withdrawal (F), mean ± SD of triplicate experiments. (G, H) Representative images of invasion (G) assays for cells under androgen stimulation or withdrawal. Scale bars, 100 µm. The number of cells in invasion (H) assays. ± s.e.m. from three biological replicates, *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t test. NS, Non significance.

Fig 3: Vitamin C reduces senescence induced by over-expression of unphosphorylatable Rb allele in vitro Both phosphorylated pRb and pRb?K7 do not co-precipitate with E2F1. Protein lysates from Rb?K7 or control wild-type thymocytes were immunoprecipitated with antibodies for pRb, for pSer243/Thr246-pRb or for pSer605-pRb, followed by immunoblotting for pRb or E2F1. Anti-pRb antibody efficiently co-immunoprecipitated E2F1 in control and Rb?K7 cells (lanes 2–3, 11), whereas the two phospho-pRb antibodies failed to co-immunoprecipitate E2F1 in either cell (lanes 7–10).Left, top, effect of retrovirus-mediated transduction of under-phosphorylated Rb?K11 (rRb?K11) on Rb-deficient mammary tumor cell proliferation as determined by MTT assay (n = 4–5 technical replicates). Left, bottom, quantification of SA-ßGAL in rRb?K11-transduced cells. Bars represent mean ± SD; P value calculated by two-tailed unpaired Student’s t-test. Right, representative images of SA-ßGAL staining showing robust senescence (arrows) in rRb?K11-transduced cells. Original magnification: 200×.Schematic diagram illustrating how unphosphorylatable Rb?K11 allele or control wild-type Rb DNA was introduced in vivo (top) or ex vivo (bottom) into retinal progenitors. i.v.c., in vitro culture.Suppression of retinal progenitor division in vivo (grey bars) and ex vivo (black bars) by Rb?K11 and p16 but not by wild-type pRb. Shown are quantification of BrdU+;GFP+ cells relative to all GFP+ cells in samples transfected with the indicated vectors (bars represent mean ± SD; ** denotes P < 0.01; ***P < 0.001 by one-way ANOVA and Tukey’s multiple comparison analysis, n = 3 biological replicates).Representative images illustrating the effect of indicated plasmids. NBL: neuroblastic layer, GCL: ganglion cell layer; scale bar: 10 µm. Arrows point to yellow cells that are Green+ and BrdU+.Vitamin C reduces cellular senescence induced by retroviral-mediated over-expression of Rb?K11. Top—images of SA-ßGAL staining in human diploid cells, IMR90, transduced with control or rRb?K11 virus with or without vitamin C added at the time of infection. Arrows point to SA-ßGAL-positive cells. Bottom left, quantification of SA-ßGAL staining (bars represent mean ± SEM; n = 4 biological replicates). P values by one-way ANOVA, Tukey's multiple comparisons test. Bottom right, dot-blot analysis with antibody for 5hmC on DNA extracted from IMR90 cells treated or not with vitamin C. Original magnification: 200×.A schematic model for the induction of DDR, senescence and diabetes in Rb?K7 mice and the effect of vitamin C diet. Normal pancreatic ß-cells can re-enter the cell cycle in response to mitogenic signals. In contrast, Rb?K7 ß-cells display a repressive chromatin and stable differentiation state, which, in response to mitogenic signals, elicits a DDR, cellular senescence, and SASP, leading to gradual islet degeneration. We propose that the epigenetic modifier, vitamin C, prevents the establishment of a stable repressive state during differentiation of Rb?K7 ß-cells, thus reduces DDR and senescence in response to mitogens, facilitating cell cycle re-entry/regeneration and alleviating diabetes in Rb?K7 mice. SC denotes senescent cell. Source data are available online for this figure.

Fig 4: Synergistic co-overexpression of TERT, E2F1, and MYC correlated with escalated tumor malignancy. (A–D) Synergistic triple high expression of TERT, E2F1, and MYC was associated with poorer overall survival (A), higher risks for biochemical relapse (B, C), and poorer metastatic-free survival (D) in PCa patients. (E–K) Proportion of PCa patients with biochemical relapse (E), advanced tumor stage (F), bone metastasis (G), higher PSA levels (H), and higher Gleason Score (I–K) was significantly higher in the group with triple high (+) expression of TERT, E2F1, and MYC. (L) Multivariate Cox regression analysis examined the synergistic effect of co-overexpression of TERT, E2F1, and MYC together with other clinicopathological features on the risk of BCR-free survival in PCa patients in TCGA cohort.

Fig 5: Effect of rs2583669 genotype on PCa patient prognosis. (A, B) Kaplan–Meier plots demonstrating the biochemical recurrence-free survival of PCa patients grouped by the genotype of rs2853669. Patients carrying C allele of rs2853669 correlated with increased risk for biochemical recurrence in the Stockholm cohort (A). Patient group with high E2F1 expression and carrying rs2853669 CC genotype underwent a higher biochemical recurrence risk (B). (C, D) PCa patient group carrying rs2853669 C allele with higher E2F1 expression tumors or T allele with higher MYC expression levels indicated an increased risk of overall survival. p-values were examined by a log-rank test.