Hypophosphorylated pRb knock-in mice exhibit hallmarks of aging and vitamin C-preventable diabetes (2021)
Figure 7

Figure 7. Vitamin C reduces senescence induced by over-expression of unphosphorylatable Rb allele in vitro

A. Both phosphorylated pRb and pRb∆K7 do not co-precipitate with E2F1. Protein lysates from Rb∆K7 or control wildtype 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).

B. 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-βGAL 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-βGAL staining showing robust senescence (arrows) in rRb∆K11-transduced cells. Original magnification: 200x.

C. Schematic diagram illustrating how unphosphorylatable Rb∆K11 allele or control wildtype Rb DNA was introduced in vivo (top) or ex vivo (bottom) into retinal progenitors. i.v.c., in vitro culture.

D. Suppression of retinal progenitor division in vivo (grey bars) and ex vivo (black bars) by Rb∆K11 and p16 but not by wildtype 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).

E. 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+.

F. Vitamin C reduces cellular senescence induced by retroviral-mediated over-expression of Rb∆K11. Top - images of SA-βGAL 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-βGAL-positive cells. Bottom left, quantification of SA-βGAL 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: 200x.

G. A schematic model for the induction of DDR, senescence and diabetes in Rb∆K7 mice and the effect of vitamin C diet. Normal pancreatic β-cells can re-enter the cell cycle in response to mitogenic signals. In contrast, Rb∆K7 β-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 β-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.

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