Extended Data Fig. 12. Analysis of the CD36 role in SASP production. Effects of CD36 inhibition.
a) Subnetwork of significant Cd36 upstream and downstream signalling interactions pulled out from FunRes global signalling interaction network for Sen SCs population at 3 d.p.i. Green nodes are related to NF-κB cascade, orange to MAPK signalling and violet to interferon regulatory factors (IRFs). b) C2C12 cells were treated with etoposide to induce cellular senescence and harvested at the indicated time points. Graphs show relative mRNA expression levels of Cd36 and SASP-related genes normalized to untreated C2C12 cells at different times after etoposide treatment (n = 3 experiments). c) TA muscles of young mice were subjected to CTX injury and mice were treated with control IgA or anti-CD36 antibody from 3 to 7 d.p.i. once per day. Representative images of MYH3 and Sirius Red staining and quantification of SA-β-gal+ cells (n = 8 TA from 7 mice for IgA, 6 TA from 4 mice for anti-CD3610μg, and 8 TA from 4 mice for anti-CD3620μg), mean CSA (n = 11 TA from 7 mice for IgA, 8 TA from 4 mice for anti-CD3610μg, and 6 TA from 4 mice for anti-CD3620μg) and frequency distribution analysis of MYH3+ fibres (n = 7 mice for IgA, 4 mice for anti-CD3610μg, and 4 mice for anti-CD3620μg) and Sirius Red staining (n = 10 TA from 6 mice for IgA, 8 TA from 4 mice for anti-CD3610μg and anti-CD3620μg) in muscle cryosections. d) Freshly sorted SPiDER+ cells from IgA or anti-CD36 antibody-treated old mice at 7 d.p.i. were cultured for 24 h in serum-deprived media, conditioned media was collected and protein levels were assessed by cytokine array. Quantification showing the proteins whose levels were reduced by 30% in the presence of anti-CD36 antibody (n = pool of 4 mice/group). e) Cytokine array analysis of whole muscle secretome from p16-3MR mice treated with (top) IgA or anti-CD36 antibody or (bottom) GCV or vehicle, as indicated before (n = pool of 3 mice for anti-CD36 and pool of 2 mice for the rest of the groups). Graphs represent the top 10 proteins whose levels were decreased in the comparison. f) Representative pictures of MYH3 and Sirius Red staining of regenerating TA muscles from IgA or anti-CD36 antibody-treated old mice at 7 d.p.i. (related to Fig. 6f). g) As in c, mRNA expression levels of the indicated genes by RT-qPCR (n = 6 TA from 3 mice in Il6 and Ifng for anti-CD36, 6 TA from 4 mice in Tnf and Ccl2 for anti-CD36, 7 TA from 4 mice in Il1b, Il12, Il18 for anti-CD36 and Ccl2 and Il12 for IgA and 8 TA from 4 mice for the rest of the genes and groups). h) EDL muscles of old mice were injured with CTX and mice were treated with IgA or anti-CD36 antibodies from 3 to 10 d.p.i. Maximum and specific force measurements and force-frequency curve are shown (n = 6 EDL from 3 mice for IgA Ab-treated and 5 EDL from 4 mice for anti-CD36-treated). Scale bars 50 μm. Results are displayed as mean ± s.e.m.; P values were calculated by Dunnet’s test (b), Tukey’s test (c), two-way ANOVA (h, force-frequency curve) and Mann–Whitney U-test (g and h).