Research Paper Volume 15, Issue 21 pp 12618—12632

Figure 3. c-Src signaling is involved in HMGB1-mediated Pax-7 expression and muscle differentiation. Western blot analysis of c-Src phosphorylation in (A) C2C12 cells treated with HMGB1 (10 ng/mL) following a time-dependent manner (n = 3) and (B) C2C12 cells pre-treated with various siRNAs against RAGE, TLR2, and TLR4 molecules (100 nM) and then exposed to HMGB1 (10 ng/mL) for 24 h. (C) qRT-PCR (n = 3) and (D) western blot analysis of Pax-7 mRNA and protein expression, respectively, in C2C12 cells after HMGB1 co-treatment with c-Src siRNA (100 nM) or c-Src inhibitor (PP2; 1 μM) for 24 h (n = 3). (E) Immunofluorescence staining to visualize the expression of Pax-7 and desmin in C2C12 cells in DM after HMGB1 (10 ng/mL) co-treatment with c-Src siRNA (100 nM) or PP2 (1 μM) for 24 h (n = 3). Pax-7 is indicated by red coloring, desmin is indicated by green, and DAPI is shown in blue. Scale bars = 100 μm. (F) Number of positively stained cells were quantified by ImageJ software (n = 3). β-Actin was used as the loading control. All data are presented as the mean ± SD of triplicate experiments. ^*p < 0.05 compared with control group; ^#p < 0.05 compared with HMGB1-treated group.