Proliferative effect of Hachimijiogan, a Japanese herbal medicine, in C2C12 skeletal muscle cells

Figures & data

Figure 1 Effects of Hachimijiogan (HJG) on C2C12 cell proliferation.

Notes: C2C12 cells were treated with various concentrations (1–200 μg/mL) of HJG and counted on Day 3. (A) Images showing C2C12 cells exposed to the indicated concentrations of HJG for 3 days. (B) Cell numbers were counted using a hemocytometer by light microscopy, and data are expressed as the percentage of the untreated control. *P<0.05 versus untreated control. (C) Cells were treated with various concentrations (1–200 μg/mL) of HJG, and an MTS assay was performed on Day 3. *P<0.05 versus untreated control.
Abbreviations: MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; OD, optical density.

Figure 2 Hachimijiogan (HJG) promotes C2C12 cell proliferation via the ERK1/2 signaling pathway.

Notes: HJG induced ERK1/2 phosphorylation in C2C12 cells. C2C12 cells were exposed to the indicated concentrations of HJG for the indicated times. After harvesting, cells were lysed and prepared for Western blotting with anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-Akt, anti-Akt, and anti-β-actin antibodies (A). The quantification of the results obtained by densitometric analysis are shown in (B) (ERK activity) and (C) (Akt activity). Values are expressed relative to the control (HJG 0). The mean of four independent experiments is presented. ****P<0.0001, ^####P<0.0001 versus untreated control (B). (D) PD98059 inhibited HJG-induced C2C12 cell proliferation. C2C12 cells were pretreated with PD98059 (50 μM) for 1 hour and then exposed to HJG with or without PD98059. MTS assays were performed on Day 3. *P<0.05 versus untreated control.
Abbreviations: min, minutes; NS, not significant; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; OD, optical density.

Figure 3 Effects of Hachimijiogan (HJG) on C2C12 cell differentiation.

Notes: C2C12 cells were induced to differentiate and treated with various concentrations (1–200 μg/mL) of HJG. To induce differentiation, the cells were grown to 80% confluence in GM and then the medium was replaced with DM consisting of DMEM with 2% heat-inactivated horse serum. (A) Cells were observed using a phase-contrast microscope. The magnification is ×100. Arrows show myotubes. (B) The numbers of multinucleated myotubes per field (1 μm² ×100) were counted using a phase-contrast microscope.
Abbreviations: GM, growth medium; DM, differentiation medium; DMEM, Dulbecco’s Modified Eagle’s Medium; NS, not significant.