## 3 Results  
Exposure to DEHP at concentrations of 1-1000 \(\upmu\)M for 96 h did not induce cytotoxicity in mouse C2C12 myoblasts under myogenic differentiation medium (Figure 1A). In fact, DEHP slightly increased cell viability at concentrations of 25 \(\upmu\)M to 1000 \(\upmu\)M (Figure 1A). Compared to DEHP, its metabolite MEHP at concentrations of 1-50 \(\upmu\)M did not affect cell viability; cell viability decreased only slightly after 100 \(\upmu\)M MEHP exposure, and displayed a greater cytotoxicity to myoblasts at concentrations of \(>\)100 \(\upmu\)M (IC50 \(\approx\) 250 \(\upmu\)M; Figure 1B). In primary HSMPCs, exposure to both DEHP and MEHP at concentrations of 1-100 \(\upmu\)M for 96 h did not induce cytotoxicity under myogenic differentiation medium (Figure 2). An increase in creatine kinase activity has been previously found in differentiating muscle cell cultures during myotube formation [25], which could be inhibited by environmental toxicants exposure [19,26]. Therefore, we next tested whether DEHP and MEHP affected creatine kinase activity. As shown in Figure 1C,D, both DEHP and MEHP at concentrationsof 25-100 uM could significantly inhibit the creatine kinase activity in differentiating C2C12 myoblasts. MEHP exposure had greater inhibitory potency than DEHP (Figure 1D). Figure 1: The changes in cell viability and creatine kinase activity in myoblasts treated with both DEHP and MEHP. Under differentiation medium, C2C12 cells treated with DEHP (**A**,**C**) or MEHP (**B**,**D**) (1–1000 uM) for 24–96 h were observed. MTT assay was performed to analyze cell viability (**A**,**B**). Creatine kinase activity was assayed using a commercial kit (**C**,**D**). GM indicated as growth medium. Data are presented as means \(\pm\) SEM (three independent experiments, triplicate repeats in each); \(p<0.05\) indicates significance compared to the control (*). Figure 2: The changes in cell viability in primary HSMPCs treated with both DEHP and MEHP. Under differentiation medium, HSMPCs treated with DEHP (**A**) or MEHP (**B**) (1–500 uM) for 24–96 h were observed. MTT assay was performed to analyze cell viability (**A**,**B**). Data are presented as means \(\pm\) SEM (three independent experiments, triplicate repeats in each). DEHP (10-100 uM) exposure effectively inhibited myotube formation in C2C12 myoblasts (\(p<0.05\)), and the responses were dose-dependent (Figure 3(Aa,Ba)). MEHP (10-50 uM) could also significantly and dose-dependently decrease myotube formation (Figure 3(Ab,Bb). Moreover, the results of immunoblot analysis show that the myogenic differentiation markers MHC and myogenin protein expression in C2C12 myoblasts were also effectively inhibited by both DEHP (Figure 4A) and MEHP (Figure 4B), and the responses were dose-dependent. Both DEHP and MEHP exposure at the concentrations of 25-100 uM also significantly decreased myotube formation (DEHP, Figure 5(Aa,Ba); MEHP, Figure 5(Ab,Bb) and protein expression of MHC and myogenin (DEHP, Figure 6A; MEHP, Figure 6B) in primary HSMPCs. Akt signaling can positively regulate skeletal myogenesis [27]. We next tested the involvement of myogenic regulatory signal Akt in myoblasts during differentiation. As shown in Figure 3, both DEHP (Figure 4A) and MEHP (Figure 4B) effectively inhibited the phosphorylation of Akt in C2C12 myoblasts (\(p<0.05\)), and the responses were dose-dependent. Both DEHP and MEHP did not affect total Akt protein expression in C2C12 myoblasts (Figure 4). Similarly, in the primary HSMPCs, both DEHP (Figure 6A) and MEHP (Figure 6B) could also effectively inhibit the phosphorylation of Akt (\(p<0.05\)); there were no changes in total Akt protein expression. Figure 3: The changes in myotube formation in C2C12 myoblasts treated with both DEHP and MEHP. Under differentiation medium, C2C12 cells treated with DEHP (**Aa,Ba**) or MEHP (**Ab,Bb**) (10–100 uM) for 96 h were observed. The cell morphology (**A**) was shown and myotube formation (**B**) was counted.