Fig 2: Modulation of apoptotic and cell cycle regulatory proteins in Ramos cells treated with HCS. (A) Ramos cells were treated with HCS (0, 12.5, 25 and 50 µl/ml) for 24 h, followed by western blot analysis to detect caspase-3, cdk2, cdk4, p21CIP1, survivin, Rb and pRb proteins. (B) Cells were treated with the indicated concentrations of HCS with or without the caspase inhibitor, ZDEVD, for 24 h and immunoblotted with the caspase-3 antibodies. (C) The effect of ZDEVD on HCS induced apoptotic cell death by Annexin V staining. ZDEVD markedly blocked the early phase of apoptosis induced by HCS while moderately modulated the late phase of apoptotic or necrotic cells (top, bar graph of early phase apoptosis; bottom, histogram). Each experiment was performed in duplicate and repeated twice independently. Data are presented as mean ± SD.

Fig 3: Protein expression following AAV2-shE6E7 transduction. E6, E7, p53, p21, pRb, Rb and p16 protein levels were measured by western blot analysis in the 3 cervical cancer cell lines 48 h following AAV2-shE6E7 or AAV-shNC transduction.

Fig 4: Diabetes in Rb?K7 mice is associated with failure of pancreatic ß-cells to re-enter the cell cycle Insulin tolerance test (ITT) demonstrating that hyperglycemic Rb?K7 mice are insulin-sensitive. Blood glucose in fasting Rb?K7 mice (n = 6) vs. control littermates (n = 6) following insulin injection. Error bars represent SD.Glucose tolerance test (GTT) demonstrating progressive defect in clearance of blood glucose in Rb?K7 mice. 6–7 week (left) or 6-month (right)-old fasting Rb?K7 mice and control littermates (n = 5–6 each per group) were injected with glucose, and blood glucose was determined at indicated intervals. Error bars represent SD.Glucose-stimulated insulin secretion (GSIS) analysis demonstrating near-normal insulin secretion in response to glucose injection in young Rb?K7 mice. Serum insulin levels in 5- to 6-week-old fasting Rb?K7 vs. control mice (n = 4 each) after glucose injection determined by ELISA. P values were calculated by two-tailed unpaired Student’s t-test. Bars represent mean ± SD.Rb?K7 mice exhibit a progressive loss of pancreatic islet mass. Left to right, representative images of pancreatic islets of 1-year-old Rb?K7 vs. control mice stained for insulin (green). Note the enlarged, irregular shape, and reduced number of pancreatic insulin+ ß-cells in Rb?K7 islets; Ratio of ß-cell area to pancreatic area in 1-month and 1-year-old Rb?K7 vs. control mice determined by immunostaining for insulin followed by ImageJ analysis; relative length of pancreatic insulin+ ß-cells in 9-month-old Rb?K7 islets (n = 5) vs. control (n = 4) using > 8 determination per biological replicate, normalized for control length. P values calculated by two-tailed unpaired Student’s t-test. Scale bar, 100 µm.Reduced levels of circulating insulin in fasting 6- to 10-month-old diabetic Rb?K7 mice compared with control wild-type littermates (0.13 vs. 0.2 ng/ml; n = 8 for each group). P value was calculated by one-tailed unpaired Student’s t-test. Bars represent mean ± SE. Average levels of blood glucose are indicated (17 mM in Rb?K7 compared with 3.2 in control mice; P = 0.0001 by two-tailed unpaired Student’s t-test).Loss of pancreatic islet area in Rb?K7 mice correlates with reduced proliferation and H3K36me3 histone marks, but not with increased cell death. TUNEL analysis showing apoptosis in 10-day-old Rb?K7 islets is even lower than in control littermates. Arrow points to a rare apoptotic cell in control islet. Serine800/804 phosphorylation of pRb in ß-cells from 10-day-old control but not Rb?K7 islets with a Ser-to-Ala substitution at this site. BrdU incorporation and PH3 staining showing reduced S-phase and mitotic cells, respectively, in 2- and 3-week-old Rb?K7 vs. control islets. Reduced H3K36me3 staining in pancreatic islets from 3-week old Rb?K7 mice compared to control littermates (n = 3 for each experiment). Scale bar, 100 µm.Quantification of BrdU incorporation, PH3 and H3K36me3 staining shown in panel (F). Mean ± SD, P values calculated by two-tailed unpaired Student’s t-test.Reduced ability of Rb?K7 ß-cell- to re-enter the cell cycle in response to pregnancy (top) or mitogenic (bottom) signals. Top, cell proliferation in P14.5 pregnant Rb?K7 islet ß-cell vs. control was determined by double immunostaining with BrdU and insulin (left); quantification (right). Results were normalized for the number of BrdU/Insulin-positive cells in control islets. An independent experiment using ki67 to quantify cell cycle re-entry is shown in Fig EV2A. Bottom, cell proliferation in Rb?K7 ß-cell vs. control islets following Exendin4 treatment was determined by immunostaining for Ki67; results were normalized for the number of positive cells in control islets. Mean ± SD, P values by two-tailed unpaired Student’s t-test. Scale bar, 100 µm.Representative immunostaining showing induction of PDX1 (islet), SOX9 (pancreatic epithelium), and MCM4 (islet) expression in Rb?K7 vs. control mice. See also Appendix Fig S5. Scale bar, 100 µm.

Fig 5: 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.