Research Paper Volume 15, Issue 21 pp 12618—12632

Figure 4. miR-342-5p/Pax-7 axis is involved in HMGB1-induced inhibition of skeletal muscle differentiation. (A) Potential miRNA targets of Pax-7 predicted by miRWalk, miRDB, and TargetScan. (B) qRT-PCR assays showing the expression of the 12 predicted miRNAs following HMGB1 treatment (10 ng/mL) (n = 3). (C) qRT-PCR assays showing the expression of miR-185-3p, miR-342-5p, and miR-499-3p in C2C12 cells after HMGB1 treatment (n = 6). (D) qRT-PCR assays showing miR-342-5p expression in C2C12 cells transfected with various siRNAs or pre-treated with c-Src inhibitor (PP2; 1 μM) prior to HMGB1 treatment for 24 h (n = 3). (E) qRT-PCR analysis and (F) western blot analysis showing the effect of miR-342-5p inhibitor on Pax-7 mRNA expression and protein expression (n = 3), respectively. (G) Immunofluorescence staining showing the expression of Pax-7 and desmin in C2C12 cells in DM after HMGB1 treatment (10 ng/mL) with 50 nM of miR-342-5p inhibitor or negative control (n = 3). Pax-7 is indicated by red coloring, desmin is indicated by green, and DAPI is shown in blue. Scale bars = 100 μm. (H) 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.