Extended Data Figure 1: The reduced autophagy flux in quiescent satellite cells can be increased by pharmacological treatment in vivo.

a, Venn diagrams of overlapping genes between a proteostasis gene set (See Supplementary Table 1) and genes significantly upregulated in quiescent satellite cells from the indicated publications or from our gene expression microarray data comparing freshly FACS isolated satellite cells from resting muscle, or muscles obtained 72 h after cardiotoxin (CTX) injury, from young, wild-type mice. b, K-means clustering analysis (performed with Gene-E, Broad Institute) of the gene expression of the autophagy-related genes during ageing. Clusters are shown with heat maps of the normalized raw data. Each column represents a different sample and each row a different gene probe. Red, increased expression; white, neutral expression; blue, decreased expression. c, Representative example of the FACS strategy and gating scheme for isolating satellite cells from mice in resting conditions. d, Pax7 and GFP immunostaining of freshly isolated satellite cells from resting muscles of young and old GFP–LC3 mice. Scale bar, 5 μm. e, Electron microscopy images of young and old satellite cells on sections of resting tibialis anterior (TA) muscle of wild-type (WT) mice. Arrowheads indicate autophagic vesicles. Scale bars, 1 μm and 0.5 μm (right and left, respectively). f, Pax7 and GFP immunostaining on tissue sections from resting tibialis anterior muscles of young and old GFP–LC3 mice. Arrowheads indicate autophagic vesicles. Scale bar, 5 μm. g, p62 and ubiquitin (Ub) MFI. Arrowheads, co-localization of p62 and ubiquitin aggregates. h, LC3 western blot of freshly isolated satellite cells from young and old, wild-type mice, treated with bafilomycin or vehicle for 4 h before collection. Graph shows LC3II quantification, after normalization with tubulin levels; for full scan see Supplementary Fig. 2. i, Quiescent satellite cells were freshly isolated from old, wild-type mice subjected to two weeks of rapamycin, spermidine or vehicle (control) treatment. Cells were treated (or not treated) with bafilomycin 4 h prior to analysis by immunostaining of LC3 marker. Z projections of representative fluorescence microscopy images are shown. Scale bars, 5 μm. j, Representative fluorescent microscopy images from Fig. 1d. Scale bar, 5 μm. Data show mean ± s.e.m. Comparisons by two-sided Mann–Whitney U-test. P values are indicated. Number of samples were n = 3 animals per group for a and b; n = 35 (young) and 66 (old) cells analysed from 3 animals for g; n = 3 animals per group for h.