Research Paper Volume 15, Issue 21 pp 12618—12632

Figure 2. RAGE, TLR2, and TLR4 receptors are involved in the downregulation of Pax-7 expression by HMGB1. C2C12 cells were pre-treated with 100 nM siRNA against RAGE, TLR2, or TLR4 for 24 h and then subjected to HMGB1 (10 ng/mL) stimulation for 24 h. The expression levels of Pax-7 mRNA and protein were respectively analyzed by (A) qRT-PCR (n = 3) and (B) western blotting assay (n = 3). (C) Immunofluorescence staining was used to visualize the expression of Pax-7 and desmin in C2C12 cells in DM after HMGB1 treatment (10 ng/mL) with indicated siRNAs (n = 3). Pax-7 is indicated by red coloring, desmin is indicated by green, and DAPI is shown in blue. Scale bars = 100 μm. (D) 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.