Fig. 5: TNC activates EGFR kinase cascade to promote MuSC proliferation.

a Coomassie blue staining of the purified recombinant mouse TNC (1–700 amino acids). b Immunoblotting analysis of the GST pull-down products with the anti-EGFR antibody. 500 ng/mL GST-tagged TNC (1–700aa) was used as the bait to incubate with MuSCs, and subsequently performed GST pull-down assay as described in Materials and Methods. Experiments were repeated independently for more than three times. c Immunoblotting analysis of EGFR activation in MuSCs. NCM was derived from both the control (sg-control) and Tnc knockout (sg-Tnc) C2C12-Mlkl-TetON cells. MuSCs were treated with combinations of 50 ng/mL EGF, 500 ng/mL GST-TNC-1-700, and 10 μM EGFR inhibitor in F-10 or NCM as indicated for 4 h. Whole cell lysates were subjected to SDS-PAGE and immunoblotting analysis using antibodies as indicated. HSP70 serves as the loading control. Experiments were repeated independently for more than three times. EGFR inhibitor: Afatinib. d Immunoblotting analysis of EGFR pathway activation in MuSCs. MuSCs were cultured in different conditions for 48 h. Whole cell lysates were subjected to SDS-PAGE and immunoblotting analysis using antibodies as indicated. F-10, F-10 medium; sg-ctl NCM, NCM derived from the control C2C12-Mlkl-TetON cells; sg-Tnc NCM, NCM derived from the Tnc knockout C2C12-Mlkl-TetON cells; NCM + EGFRi, normal NCM with EGFR inhibitor (10 μM Afatinib). HSP70 serves as the loading control. Experiments were repeated independently for more than three times. e Quantification of MuSCs that were cultured in NCM with TNC or EGFR neutralizing antibodies. MuSCs were cultured for 48 h. 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. NCM + α-TNC, NCM with antibody against TNC to neutralize TNC; NCM + α-EGFR, NCM with antibody against EGFR to block EGFR activation. The antibodies were titrated to different doses as indicated. f Quantification of Egfr knockout MuSCs that were cultured in NCM. MuSCs were isolated from Cas9-expressing mice and infected by AAV encoding sgRNA against Egfr (sg-Egfr) or the scramble control (sg-control). The Egfr and the control knockout MuSCs were cultured in F10 or NCM for 48 h. 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. g Immunoblotting analysis of the knockout efficiency of EGFR in AAV-infected MuSCs. The asterisk (*) denotes the non-specific band. h Representative immunofluorescence staining of p-EGFR (green) and Pax7 (red) in freshly isolated MuSCs (2 days after CTX injection). MuSCs were isolated by FACS and fixed on PDL/Collagen-I pre-coated coverslip, as described in Materials and Methods, followed by immunofluorescence staining using the antibodies as indicated. Nuclei were identified by staining with DAPI. Scale bars, 5 μm. i Quantification of the p-EGFR⁺/Pax7⁺ cells as shown in h. MuSCs isolated from 3 mice were pooled together for immunofluorescence staining in each group. The histogram represents the percentage of p-EGFR⁺/Pax7⁺ cells out of 200 Pax7⁺ cells per genotype. The data are expressed as the means ± SD of 6 images. j Representative H&E staining of TA muscle cross sections from injured Rag2^−/−;Il2rg^−/− mice with antibodies neutralizing TNC/EGFR activation. After CTX injection, individual anti-TNC, anti-EGFR, or anti-EGF antibody was injected intramuscularly every other day as described in Materials and Methods. Twelve days later, TA samples were harvest and prepared for H&E staining. Scale bars, 40 μm. k Quantification of myofiber sizes from cross-sectional areas as representative shown in j. 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. The data are expressed as the means ± SD. n = 6 for each group of mice. P values for e, f, and k, were determined by one-way ANOVA with Tukey’s multiple comparisons test; P value for i was determined by unpaired two-tailed t-test with Welch’s correction. ns, non-significant; *P < 0.05; ***P < 0.005.