Fig 2: TNC was released by necroptotic myofibers to promote MuSC proliferation.a Procedures for purification of the MuSC proliferation-promoting factor(s) from NCM. b Representative phase-contrast images of MuSCs cultured in different fractions of NCM after purification. The complete NCM was used as the positive control (NCM input); C2C12-Mlkl-TetON medium without tetracycline treatment was used as the negative control (Tet- control). Cells were cultured for 48 h. Scale bars, 100 µm. c Quantification of MuSCs that were expanded in different fractions of NCM after purification. Cell proliferation was determined by measuring intracellular ATP levels using CellTiter-Glo assay. The data are expressed as the means ± SD of 3 technical repeats. d Identification of TNC as a major effector in the active fraction of NCM. After purification, 8 µg proteins from each fraction were subjected to SDS-PAGE separation, followed by silver staining. The indicated bands were excised and subjected to mass spectrometry analysis. Protein IDs were shown as indicated. The red arrow indicates the position of TNC. e Immunoblotting analysis of TNC in ACM and NCM. 10 mL of ACM or NCM were harvested and concentrated using ultra centrifugal filters then subjected to run SDS-PAGE and followed by immunoblotting analysis. IGF1 was used as the loading control. The asterisk (*) denotes the non-specific band. Experiments were repeated independently for more than three times. f The knockout-rescue strategy of Tnc in C2C12-Mlkl-TetON cell line. g Immunoblotting analysis of TNC expression in Tnc knockout and rescued C2C12-Mlkl-TetON cell lines. GAPDH serves as the loading control. h Quantification of MuSCs cultured in NCM with or without TNC expressing. NCM was derived from Tnc knockout or rescued C2C12-Mlkl-TetON cells, as shown in g. MuSCs were cultured for 3 passages followed by cell proliferation analysis, which was determined by measuring intracellular ATP levels. The data are expressed as the means ± SD of 3 technical repeats. i Immunoblotting analysis of the expression and extracellular release of TNC upon necroptosis induction at different time points as indicated. C2C12-Mlkl-TetON cells were treated with 1 µg/mL tetracycline for 12 h to induce necroptosis. Both the whole cell lysates and NCM were harvested and then subjected to immunoblotting analysis using antibodies as indicated. Extracellular release of GAPDH was used as a control to indicate membrane disruption caused by necroptosis; IGF1 was used as the loading control for NCM; HSP70 serves as the loading control for the whole cell lysates. Experiments were repeated independently for more than three times. j Representative immunohistochemical staining of TNC in TA muscle cross sections from injured mice (0, 2 and 3 days after CTX injection as indicated). The signals of TNC appear brown in sections counter-stained with hematoxylin (blue). Scale bars, 50 µm. k Quantification of the MuSCs in injured TA muscles (3 days after CTX injection) of Tncf/f and MCK-Cre:Tncf/f mice by FACS analysis. MuSCs were isolated as described in Materials and Methods. Histogram represents the percentage of MuSCs (7-AAD-CD11b-CD31-CD45-Sca1-Vcam+ population, and 7-AAD-CD11b-CD31-CD45-Sca1-Vcam+Integrin-a7+ population) out of the total digested mono-nucleus cells. 7-AAD reagent was used to exclude dead cells. n = 4 mice for each genotype. The data are expressed as the means ± SD. l Representative H&E staining of TA muscle cross-sections from uninjured or injured (7 days after CTX injection) Tncf/f and MCK-Cre:Tncf/f mice. Scale bars, 50 µm. m Quantification of myofiber sizes from CSAs of injured mice 7 days after CTX injection. Histogram graph represents averaged myofiber size. The sizes of each 900 adjacent regenerating myofibers with central nuclei were measured for every mouse. Each dot represents an individual mouse. n = 4 mice for each genotype. The data are expressed as the means ± SD. P values for c and h were determined by one-way ANOVA with Tukey’s multiple comparisons test; P values for k and m were determined by unpaired two-tailed t-test; ns, non-significant; ***P < 0.005.