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. 2018 May 21;2018:6463807. doi: 10.1155/2018/6463807

Figure 9.

Figure 9

Effect of nonsterol isoprenoids and soluble cholesterol treatments on myotube index (MI) in C2C12 myoblasts affected by statins or MβCD. C2C12 myoblasts were exposed for 24, 72, or 120 h to statins or MβCD (IC50) (day 1—proliferating myoblasts, day 3—differentiating myotubes, and day 5—differentiated myotubes). ATR or SIM was administered in the listed concentration at each day of differentiation: ATR: day 1—76 μM, day 3—46 μM, and day 5—36 μM; SIM: day 1—87 μM, day 3—6 μM, and day 5—3 μM. MβCD was added to give the final concentration: day 1—2.7 mM, day 3—1.9 mM, and day 5–1.1 mM or vehicle control (0.1% DMSO or 2% HS DMEM) without or with selected mevalonate pathway intermediate (mevalonate 100 μM, geranylgeraniol 10 μM, farnesol 10 μM, Chol-PEG 1 mM, dolichol 1 μg/mL, and ubiquinol 10 μg/mL). Next, cells were subjected to immunofluorescence with HO33342 to counterstain nuclei (see Materials and Methods). The myotube index was determined as the ratio of the nuclear number in myotubes (C2C12 cells with three or more nuclei) to the total number of nuclei multiplied by 100%). (a) Atorvastatin (ATR, IC50) did not affect myotube index (MI) at days 1 and 3. However, it significantly diminished fraction of myonuclei in myotubes at day 5. Neither of added nonsterol isoprenoids nor cholesterol treatment could influence ATR effect. (b) Simvastatin (SIM, IC50) did not change MI at day 1, but it significantly lessened myotube representation at days 3 and 5. Nonsterol isoprenoids and cholesterol reversed SIM effect at day 3 but not at day 5. (c) Methyl-beta-cyclodextrin (MβCD, IC50) could not effect MI at day 1, but it significantly decreased percentage of myotubes at days 3 and 5 without any effect of nonsterol isoprenoids or cholesterol. Two-way ANOVA test for MI followed by Bonferroni's multiple comparisons was employed to analyze the data. The results of [time (proliferating myoblasts, differentiating myotubes, differentiated myotubes)] amounted to F(2,337) = 80.99, p < 0.0001 for ATR; F(2,352) = 98.91, p < 0.0001 for SIM; and F(2,266) = 59.00, p < 0.0001 for MβCD. Treatment: ATR, ATR + MEV, ATR + GGOH, ATR + FOH, and ATR + Chol-PEG (F(6,337) = 5.982, p < 0.0001); SIM, SIM + MEV, SIM + GGOH, SIM + FOH, and SIM + Chol-PEG (F(6,352) = 6.370, p < 0.0001); MβCD, MβCD + MEV, MβCD + GGOH, MβCD + FOH, and MβCD + Chol-PEG (F(5,266) = 14.77, p < 0.0001). Interaction: F(12,337) = 3.096, p = 0.0004 for ATR; F(12,352) = 0.8962, p = 0.5511 for SIM; F(10,266) = 2.560, p = 0.0057 for MβCD. Error bars = SEM and ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 for comparison between the means. Results are means of three independent experiments.