Figure 1.
Maduramicin inhibits cell growth in myoblast cells.
C2C12 and RD cells (plated in triplicates) were exposed to maduramicin at indicated concentrations for 5 days, followed by cell counting (A) and morphological analysis (B). Data represents mean ± SE (n = 3, corresponding to three independent experiments). **P<0.01, difference with the control group.
Figure 2.
Maduramicin inhibits cell proliferation in myoblast cells.
C2C12 and RD cells (plated in triplicates) were exposed to maduramicin at indicated concentrations for 24, 48 or 72 h, followed by one solution assay. Data represents mean ± SE (n = 6, corresponding to six independent experiments). *P<0.05, **P<0.01, difference with the control group.
Figure 3.
Maduramicin induces cell death in myoblast cells.
C2C12 and RD cells (plated in triplicates) were exposed to maduramicin at indicated concentrations for 24, 48 or 72 h, followed by trypan blue exclusion assay. Data represents mean ± SE (n = 3, corresponding to three independent experiments). *P<0.05, **P<0.01, difference with the control group.
Figure 4.
Maduramicin arrests C2C12 cells at G0/G1 phase of the cell cycle.
C2C12 cells were treated with maduramicin for 24 h at indicated concentrations (A), or for indicated time at 0.5 µg/ml (B, C), followed by staining with PI and flow cytometry. (A, B) Results are presented as means ± SE (n = 3, corresponding to three independent experiments). *P<0.05, **P<0.01, difference with the control group. (C) Histograms from a representative experiment show the time-course effect of maduramicin on cell cycle profile in C2C12 cells. Note: Maduramicin increased sub-G1 in a time-dependent manner.
Figure 5.
Maduramicin downregulates protein expression of cyclin D1, CDK4, CDK6, and CDC25A, and upregulates expression of p21Cip1 and p27Kip1, resulting in hypophosphorylation of Rb in C2C12 cells.
C2C12 cells were treated with maduramicin for 24 h at indicated concentrations, followed by Western blotting with indicated antibodies. β-Tubulin was used for loading control. Representative blots are shown (A). Blots for indicated proteins were semi-quantified using NIH image J (B). Results are presented as means ± SE (n = 3, corresponding to three independent experiments). *P<0.05, **P<0.01, difference with the control group.
Figure 6.
Maduramicin induces apoptosis in C2C12 cells.
C2C12 cells were treated with maduramicin for 72 h at indicated concentrations, followed by Annexin V-PI staining and flow cytometry. (A) Histograms from a representative experiment show the apoptotic effect of maduramicin on C2C12 cells. The percentages of necrotic, late apoptotic, viable, and early apoptotic cells are displayed in Q1, Q2, Q3 and Q4, respectively. (B) Bar graphs show that maduramicin induced apoptosis of C2C12 cells in a concentration-dependent manner. Quantitative results (Q2+Q4) are displayed as fold change compared with control. Data represents mean ± SE (n = 3, corresponding to three independent experiments). **P<0.01, difference with the control group.
Figure 7.
Maduramicin upregulates expression of DR4, TRADD, TRAIL, BAK and BAD, leading to activation of caspases 8, 9 and 3 as well as cleavage of PARP in C2C12 cells.
C2C12 cells were treated with maduramicin for 24 h at indicated concentrations, followed by Western blotting with indicated antibodies. β-Tubulin was used for loading control. Representative blots are shown (A, C and E). Blots for indicated proteins were semi-quantified using NIH image J (B, D and F). Results are presented as means ± SE (n = 3, corresponding to three independent experiments). *P<0.05, **P<0.01, difference with the control group.
Figure 8.
Maduramicin induces caspase-dependent apoptosis in C2C12 cells.
C2C12 cells (plated in triplicates), pretreated with or without z-VAD-fmk (20 µM) for 1 h, were treated with maduramicin for 24 h at indicated concentrations, followed by Western blotting with indicated antibodies (A), or for 48 h at indicated concentrations, followed by trypan blue exclusion assay (B). Data represents mean ± SE (n = 3, corresponding to three independent experiments). *P<0.05, **P<0.01, difference with the control group. #P<0.05, difference with z-VAD-fmk group.