Extended Data Figure 5: ROS inhibition in autophagy-impaired aged and Atg7 null satellite cells significantly restores cell proteostasis.

a, ROS-level quantification in quiescent satellite cells from three-month-old Atg7^WT and Atg7^ΔPax7 mice by CellROX flow cytometry. Representative images are shown. Scale bar, 5 μm. b, Western blot analysis of 53BP1 and parkin in satellite cells isolated from three-month-old Atg7^WT and Atg7^ΔPax7 mice. Tubulin control is the same tubulin control for Fig. 3g. Graph shows quantification of 53BP1 and parkin protein normalized to tubulin; for full scan see Supplementary Information Fig. 1. c, Quantification of ROS levels for satellite cells isolated from young and geriatric WT mice by flow cytometry using CellROX fluorescent dye. Satellite cells were treated with Trolox or vehicle (control) for 48 h before analysis. Results are represented as variation of MFI between young and geriatric satellite cells. d, Quantification of p62 and ubiquitin protein levels on immunostained freshly isolated satellite cells from resting muscle of old wild-type mice, in vivo treated for 2 weeks with Trolox or vehicle (control). Representative images are shown. Scale bar, 5 μm. e, Western blot analysis of LC3 and tubulin in satellite cells isolated from geriatric WT mice and treated for 48 h with Trolox or vehicle (control), in the absence or presence of bafilomycin for 4 h before analysis. Graph shows quantification of LC3II protein normalized to tubulin; for full gel scan see Supplementary Information Fig. 2. f, Autophagy flux and mitochondria in satellite cells from GFP–LC3 mice (two weeks with or without Trolox treatment). Satellite cells treated for 4 h ± bafilomycin treatment. Representative images are shown. Scale bar, 5 μm. g, The mRFP–GFP–LC3 plasmid was transfected into young or geriatric satellite cells, with 48 h treatment ± Trolox and then 4 h treatment ± bafilomycin, prior to fixation. The percentage of autophagosomes was quantified as in Fig. 2a. h, Muscle regeneration using geriatric satellite cell transplantation. An equal number of freshly isolated geriatric satellite cells, infected with GFP lentivirus and treated for 48 h with Trolox or vehicle, were transplanted into injured muscle of young immunodeficient mice. Four days later, muscles were collected and immunostained for GFP, MyoD and Mgn (to determine the possible myogenic states of satellite cells in the regenerating muscle). Representative images are shown. Scale bar, 50 μm. i, ChIP analysis for H2AK119ub (H2Aub) in satellite cells isolated from young and geriatric wild-type mice. j, Quantification of proliferating (BrdU⁺) and senescent (SA-β-gal⁺) satellite cells isolated from Atg7^WT and Atg7^ΔPax7 mice treated 48 h with Trolox or vehicle (control) and cultured for 96 h. k, Quantification of proliferating (BrdU⁺) and senescent (senescence-associated β-gal⁺) satellite cells isolated from Atg7^WT and Atg7^ΔPax7 mice and infected with LV-sh p16^INK4a or LV-sh scramble, and cultured for 96 h. Data show mean ± s.e.m. Comparisons by two-sided Mann–Whitney U-tests. P values are indicated. Number of samples were n = 60,000 cells analysed from 3 animals (a); n = 3 animals per group (b); n = 60,000 cells analysed from 3 animals (c); n = 36 (control) and n = 35 (Trolox) cells analysed from 3 animals (d); n = 3 animals per group (e); n = 60,000 cells analysed from 3 animals (f); n = 21 (young), n = 20 (young, Trolox), n = 19 (young, + bafilomycin), n = 18 (young, Trolox + bafilomycin), n = 21 (geriatric), n = 19 (geriatric, Trolox), n = 15 (geriatric, + bafilomycin) and n = 37 (geriatric, Trolox + bafilomycin) cells analysed from 3 animals (g); n = 3 animals per group (i–k).