Fig. 2: Necroptotic myofibers are indispensable for post injury MuSC proliferation.

a Representative H&E staining of TA muscle cross-sections from injured mice at different time points (0, 1, 2, 3, 5, and 7 days after CTX injection, respectively). The continuous dotted lines delineate the borders of death-resistant myofibers. Scale bars, 100 μm. b Immunoblotting analysis of RIPK1, RIPK3, MLKL, FADD, Cleaved Caspase-3, and FLIP_L expression in myofibers at different time points as indicated. Myofibers were purified from TA muscles as described in Materials and Methods. Whole myofiber lysates were exacted from 3 mice and pooled together for each condition. HSP70 serves as the loading control. Experiments were repeated independently for more than three times. c Representative immunohistochemical staining of p-MLKL in TA muscle cross sections from injured mice (2 days after CTX injection). The signals of p-MLKL appear brown in sections counter-stained with hematoxylin (blue). Experiments were repeated independently for more than three times. Scale bars, 50 μm. d Representative of H&E staining of TA muscle cross-sections from both uninjured (Day 0) and injured (7 and 15 days after CTX injection) Mlkl^f/f and MCK-Cre:Mlkl^f/f mice. Scale bars, 50 μm. e Quantification of myofiber sizes from cross-sectional areas (CSAs) of injured mice (7 days after CTX injection) and myofibers with multiple central nuclei out of total cells with central nuclei (in vivo fusion index) 15 days post CTX injection. 3 different views were counted for each mouse. The data are expressed as the means ± SD. n = 6 each for Mlkl^f/f and MCK-Cre:Mlkl^f/f mice. f Representative H&E staining of TA muscle cross-sections from injured (7 days after CTX injection) Mlkl^f/f and MCK-Cre:Mlkl^f/f mice. The continuous dotted line delineates the borders of cell death-resistant myofibers. Scale bars, 100 μm. g Representative immunohistochemical staining of p-MLKL in TA muscle cross sections from injured (2 days after CTX injection) Mlkl^f/f and MCK-Cre:Mlkl^f/f mice. The signals of p-MLKL appear brown in sections counter-stained with hematoxylin (blue). Experiments were repeated independently for more than three times. Scale bars, 50 μm. h Quantification of the MuSCs in injured TA muscles (3 days after CTX injection) by FACS analysis. MuSCs were isolated as described in Materials and Methods. Histogram represents percentage of MuSCs (PI^-CD11b^-CD31^-CD45^-Sca1^-Vcam⁺ population, and 7-AAD^-CD11b^-CD31^-CD45^-Sca1^-Vcam⁺Integrin-α7⁺ population) out of the total digested mono-nucleus cells. The data are expressed as the means ± SD. Left panel: n = 6 each for Mlkl^f/f and MCK-Cre:Mlkl^f/f mice. Right panel: n = 4 for Mlkl^f/f and n = 5 for MCK-Cre:Mlkl^f/f mice. i qRT-PCR analysis of Ccnd1 mRNA level in MuSCs isolated from injured mice (3 days after CTX injection). The mRNA level of Gapdh was used as the internal control. MuSCs isolated from 3 mice were pooled together for qRT-PCR analysis. The data are expressed as the means ± SD of 3 technical repeats. P values for e and i were determined by unpaired two-tailed t-test; P values for h were determined by unpaired two-tailed t-test with Welch’s correction. *P < 0.05; ***P < 0.005.