Fig. 3: Necroptotic muscle cells release factors to promote MuSC proliferation.

a Schematics of generating stable C2C12 cell lines that are capable of Tet-induced necroptosis or apoptosis, through overexpressing MLKL or tBid, respectively. b Immunoblotting analysis of MLKL-Flag, tBid-Flag, and Cleaved Caspase-3 expression in cell death-inducible C2C12 cell lines with or without tetracycline treatment. C2C12-Mlkl-TetON cells were treated with 1 μg/mL tetracycline for 12 h to induce necroptosis, and C2C12-tBid-TetON cells were treated with 1 μg/mL tetracycline for 6 h to induce apoptosis. Whole cell lysates were subjected to SDS-PAGE and immunoblotting analysis using antibodies as indicated. GAPDH serves as the loading control. The asterisk (*) denotes the non-specific band. Experiments were repeated independently for more than three times. c Schematic experimental design for the collection and application of cell death conditioned medium. d Representative phase-contrast images (left) and quantification of MuSC cell numbers (right) after 48 h culturing. 20,000 freshly isolated MuSCs were seeded in 35 mm dish and cultured with the indicated medium for 48 h. NCM necroptosis conditioned medium, ACM apoptosis conditioned medium. Scale bars, 100 μm. The data are expressed as the means ± SD of 3 independent experiments. e Proliferation analysis of NCM-cultured MuSCs by BrdU assay. Freshly isolated MuSCs were cultured in F-10 medium or NCM for 48 h, followed by 10 μM BrdU labeling for 2 h and analyzed by FACS. f Quantification of the BrdU⁺ MuSCs as representatively shown in e. The data are expressed as the means ± SD of 4 independent experiments. g Immunofluorescence staining of Pax7 (green) and MyoD (red) in F-10 or NCM-expanded MuSCs. Cells were cultured and expanded for 2 passages in the corresponding medium followed by immunofluorescence staining as described in Materials and Methods. Nuclei were identified by staining with DAPI. Scale bars, 50 μm. h Quantification of the percentages of Pax7^hiMyoD^low, Pax7^hiMyoD^hi, Pax7^lowMyoD^hi and Pax7^lowMyoD^low subpopulations of MuSCs as shown in g. Histogram represents the percentages of each subpopulation out of 300 cells per condition. Fluorescent intensity was measured by Image Pro-Plus as described in Materials and Methods. The data are expressed as the means ± SD of 6 images. i Representative immunofluorescence staining of MyHC (green) in differentiated MuSCs. Nuclei were identified by staining with DAPI. Scale bars, 50 μm. Experiments were repeated independently for more than three times. j Representative immunofluorescence staining of Laminin (green) merged with red fluorescent engrafted transplanted MuSCs. Red fluorescent MuSCs isolated from R26^mT/mG transgenic mice were expanded in F-10 medium or NCM for 2 passages and then transplanted into X-ray irradiated recipient, the injured nonfluorescent Rag2^−/−;Il2rg^−/− TA muscles. Freshly isolated MuSCs were transplanted and used as a positive control. Cross-sections of TA muscles were harvested at 28 days after transplantation and prepared for immunofluorescence staining of Laminin. Nuclei were identified by staining with DAPI. Scale bars, 25 μm. k Quantification of the engrafted Tomato⁺ myofibers as shown in j. The number of Tomato⁺ myofibers from 24 fields (Leica SP8 microscopy with 20x objective magnification) were quantified for each mouse. Each dot represents an individual mouse. The data are expressed as the means ± SD. n = 6 for each group of recipient mice. P values for d and k were determined by unpaired two-tailed t-test; P value for f was determined by unpaired two-tailed t-test with Welch’s correction; P values for h were determined by two-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.005.