Extended Data Figure 5: p16^INK4a regulates proliferation and promotes geroconversion of geriatric, progeric and Bmi1-null satellite cells in vitro and in vivo.

a, FACS-purified geriatric satellite cells were transduced with Ad-shRNAp16^INK4a or Ad-shScramble, cultured in growth medium containing high serum and FGF2 and progeny was quantified over time. b, FACS purified Bmi1^−/− satellite cells were transduced with Ad-shRNAp16^INK4a or Ad-shScramble, cultured in growth medium for 4 days, and BrdU incorporation was quantified after 1 h pulse. c, Representative pictures of eMHC immunostaining in cryosections from grafted extensor digitorum longus muscle of geriatric wild-type mice transduced (at the moment of grafting) with Ad-shRNAp16^INK4a or Ad-shScramble, 1 week after heterografting into the tibialis anterior muscle of young wild-type mice. Number and size of regenerating myofibres (eMHC⁺ fibres), and relative mRNA levels of p16^INK4a, p15^INK4b and Igfbp5 are shown. d, Ad-shRNAp16^INK4a or Ad-shScramble transduction in SAMP8 extensor digitorum longus muscle heterografts as in panel c. e, Representative pictures of Pax7 (green) immunostaining and DNA counterstained with DAPI (red) in cryosections from extensor digitorum longus muscle from geriatric mice from panel c. Arrows indicate Pax7⁺ cells. The number of proliferating (Pax7/Ki67 double-positive) satellite cells per section is shown. Scale bar, 50 μm. f, Regenerating eMHC⁺ myofibre size, p16^INK4a expression, number of proliferating (Pax7/Ki67 double-positive) satellite cells and total number of satellite cells (Pax7⁺) per section in grafted extensor digitorum longus muscle from geriatric (28-month-old) p16^INK4a/Arf^fl/fl mice, transduced (at the moment of grafting) with Ad-Cre or Ad-GFP, 1 week after heterografting onto the tibialis anterior muscle of young, 3-month-old wild-type mice. g, Plasmid vectors expressing p16^INK4a or GFP, as control, were electroporated into resting tibialis anterior muscles of young wild-type mice for incorporation into quiescent satellite cells. One day after, injury was induced by CTX injection. After 7 days, satellite cells were FACS isolated, and the number of satellite cells that had expanded from the initial quiescent stem-cell population was determined, and the expression levels of p16^INK4a, p15^INK4b and Igfbp5 were analysed by RT–qPCR. h, Number of colonies derived from single cells as a measure of the proliferative capacity and relative mRNA levels of p16^INK4a, p15^INK4b and Igfbp5 evaluated by RT–qPCR from young satellite cells isolated by FACS, transfected with plasmid vectors expressing p16^INK4a or GFP (as control) and cultured in proliferative conditions for 96 h. i, As in Fig. 4a, d, equal numbers of FACS-purified satellite cells from adult, old and geriatric mice were transplanted into muscle of young mice. Four days after transplantation, GFP⁺ satellite cells were re-isolated by FACS and analysed for the expression of Rb/E2F targets (cyclin A, cyclin E, Lmnb1 and Mcm3) and Cdc6. Scale bars, 50 μm. Data are mean ± s.e.m. Two-sided Mann–Whitney U-test was used to assess statistical significance. P values are indicated. a, b, n = 4 biological replicates; c–f, i, n = 4 donor mice; g, n = 6 biological replicates; h, n = 5 biological replicates. d, e, Two muscles grafted per donor mouse. i, At least three independent engraftments per donor mouse. n.s., not significant.