Extended Data Figure 3: p16^INK4a silencing restores reversible quiescence in geriatric and progeric satellite cells.

a, Number of activated (Pax7⁺MyoD⁺) satellite cells, and p16^INK4a, p15^INK4b and Igfbp5 expression by RT–qPCR in tibialis anterior muscle from geriatric mice transduced with Ad-shRNAp16^INK4a or Ad-shScramble, 14 h after CTX injury. Expression values are referred to adult. Similar results were obtained after p16^INK4a silencing in satellite cells of geriatric muscle through p16^INK4a shRNA (or shScramble) liposome-mediated delivery (not shown). As control, we confirmed that p16^INK4a shRNA had no effect in adult or old satellite cells (not shown). b, Scheme of transplantation assay of labelled satellite cells into pre-injured young hosts. Equal numbers of satellite cells from resting muscle of adult old and geriatric mice isolated by FACS were labelled with PKH26 dye, and transplanted into young mice (as in Fig. 2a); senescence-associated markers including p16^INK4a, p15^INK4b and Igfbp5 were analysed by RT–qPCR of 24-h-activated satellite cells. c, Scheme of transplantation assay of labelled satellite cells into pre-injured young hosts. Equal numbers of satellite cells from resting muscle of SAMP8 and Bmi1 null mice, and their corresponding age-matched control mice, were isolated by FACS, p16^INK4a-silenced via liposome-mediated delivery of p16^INK4a shRNA (or shScramble) and labelled with PKH26 dye, and immediately transplanted into pre-injured muscle of young mice; satellite cell activation was analysed 24 h later by quantifying the number of sorted MyoD⁺ or Ki67⁺ satellite cells (only results of MyoD⁺ cells are shown); senescence-associated markers including p16^INK4a, p15^INK4b and Igfbp5 were analysed in sorted labelled cells by RT–qPCR. d, Satellite cells from adult, old or geriatric mice were cultured in differentiation medium, for 96 h to obtain ‘reserve quiescent satellite cells’ (first round myogenesis); subsequently, reserve cells were subjected to a second myogenic round (reactivated with growth medium and cultured in differentiation medium for an extra 96-h period) to obtain secondary reserve quiescent satellite cells (second round myogenesis). Return to quiescence (self-renewal) was defined as Pax7⁺ reserve satellite cells that could not incorporate BrdU (BrdU negative). Alternatively, geriatric satellite cells cultured in differentiation medium were transduced with Ad-shRNAp16^INK4a or Ad-shScramble, and the number of ‘reserve quiescent cells’ was analysed in the two successive myogenesis rounds. e, After quiescence entry, as in panel d, reserve satellite cells were reactivated with growth medium for 48 h, and the increase in number of activated satellite cells (BrdU⁺ cells after 1 h pulse) was calculated compared to the control. The reactivation capacity of geriatric reserve satellite cells in vitro was assayed after adenoviral infection with Ad-shRNAp16^INK4a (or Ad-shScramble). f, Satellite cells from young mice were transduced with vectors (pBabe) expressing p16^INK4a (or GFP as control) and cultured in differentiation medium to obtain reserve satellite cells, and reactivated with growth medium as in panel d. The percentage of cells incorporating BrdU after 1-h pulse was calculated. Data are mean ± s.e.m. Two-sided Mann–Whitney U-test was used to assess statistical significance. P values are indicated. a, n = 5 donor mice; b, c, n = 4 donor mice; d, e, n = 4 biological replicates; f, n = 3 biological replicates. b, c, At least three independent engraftments per donor mouse. n.s., not significant.