Extended Data Figure 4: Autophagy loss in satellite cells causes dysfunctional mitophagy and mitochondria accumulation, leading to increased ROS and senescence.

a, p62 and ubiquitin immunostaining on freshly isolated satellite cells from resting muscle of three-month-old Atg7^WT and Atg7^ΔPax7ER mice, one month after tamoxifen treatment. Arrowheads indicate co-localization of p62 and Ub aggregates. Representative images are shown. Scale bar, 5 μm. b, TOM20 and Lamp1 immunostaining of quiescent satellite cells isolated from young and geriatric WT mice. Mice were subjected to two weeks of rapamycin, spermidine or Trolox (or vehicle) treatment before analysis. Co-localization was calculated as the area occupied by the immunofluorescence co-localizing staining on images with respect to the total cellular area. The Pearson’s coefficient (r) was used to analyse the correlation of the intensity values of green and red pixels in the dual-channel images. The z projections of representative fluorescence microscopy images are shown. Scale bar, 5 μm. c, Mitochondria quantification by MitoTracker in quiescent satellite cells of old mice, treated with rapamycin or vehicle for two weeks. d, Mitochondria (MitoTracker labelling) in young or geriatric cells. Satellite cells, were pre-treated with CCCP for 1 h (see Methods) and ± rapamycin for 24 h. Percentage of MitoTracker MFI reduction ± rapamycin. e, For the mitochondrial membrane potential analysis, satellite cells were freshly isolated from young wild-type mice and treated for 1 h with CCCP or DMSO (control). Membrane potential (TMRM MFI/MitoTracker Green MFI ratio) of cells was calculated by flow cytometry analysis at 1 h and 24 h after CCCP treatment (being 100% the membrane potential value of control satellite cells). f, Mitochondria content was quantified by MitoTracker staining of satellite cells from young and geriatric wild-type mice treated with rapamycin or vehicle (control) for 48 h. The z projections of representative fluorescence microscopy images are shown. Scale bar, 5 μm. g, Mitochondria and ROS detection by MitoTracker and CellROX staining, respectively. Co-localization was calculated as in b. The z projections of representative fluorescence microscopy images are shown. Scale bar, 5 μm. h, Representative images of freshly isolated satellite cells from resting muscle of three-month-old Atg7^WT and Atg7^ΔPax7 mice stained with CellROX fluorescent dye and p16^INK4a antibody. Scale bar, 5 μm. Data are mean ± s.e.m. Comparisons by two-sided Mann–Whitney U-test. P values are indicated. Number of samples were n = 36 (Atg7^WT) and n = 38 (Atg7^ΔPax7ER) cells analysed from 3 animals (a); n = 23 (young), n = 24 (control), n = 42 (rapamycin); n = 28 (spermidine) and n = 21 (Trolox) cells analysed from 3 animals (b); n = 60,000 cells analysed from 3 animals (c); n = 40,000 cells analysed from 4 animals (d); n = 30,000 cells analysed from 3 animals (e, f); n = 18 (young), 21 (control), 15 (rapamycin) and 13 (Trolox) cells analysed from 3 animals (g).