Extended Data Fig. 6: Senescent macrophages induce Ferroptosis of muscle cells through disruption of local iron homeostasis in the Quadriceps during OA progression.
(a) Data statistics of raw data from Quadriceps Proteomics analysis. (b) The integrated intensity of Perl’s staining (n = 13 sham, n = 15 DMM) and Turnbull’s staining (n = 5) in the Quadriceps muscle of mice at S12 and D12 was quantified. Perl’s staining was utilized for the measurement of ferric iron content, while Turnbull’s staining was employed to assess ferrous iron content. (c) The top enriched terms of GO analysis for significant DEPs of Quadriceps in mice between D12 vs S12. (d) The strategies and representative profiles of flow cytometry analysis for indicted dyes staining on mitochondria in Quadriceps of mice at S12 and D12. FMO: Fluorescence Minus One. (e) Blots and quantitative results of iron metabolism regulators in quadriceps of mice at S12 and D12 (n = 3). (f) Representative FerroOrange staining images and MFI analysis of macrophages from the Quadriceps of mice at S12 and D12. (n = 5). Scale bar=5 μm. (g) The flow cytometry strategy, representative images, and quantitative results of Fe2+ Dye intensity in macrophage from Quadriceps of mice at S12 and D12, (n = 7 Sham, n = 6 DMM). (h–j) Representative flow cytometry profiles and MFI of indicated dye staining on C2C12 cells stimulating with the conditional medium of macrophages (n = 5). (k) The percentage of dead C2C12 cells under conditional medium stimulation with or without Deferoxamine-B treatment. PI dye staining was employed to identify dead cells (n = 5). (l-m) The MFI of C11 BODIPY 581/591 staining on C2C12 cells under conditional medium stimulation with or without (l) Gpx4 and (m) Fsp1 over-expression. (n = 5). Data are presented as mean±s.e.m. unless otherwise noted, all statistical tests were two-sided unless otherwise noted, and n represented biological replicates unless otherwise noted. Student’s t-test (b, d, e, f, g, h, i, j). One-way ANOVA with Bonferroni test (k). One-way ANOVA with Tamhane’s T2 test (m). Kruskal- Wallis test (l). median (25% IQR, 75% IQR) (l).
