Fig. 3: Kinesin-1 controls dexamethasone-promoted myogenic differentiation.

a Total cell lysate from C2C12 cells expressing non-silenced (shCon) or Kinesin-1-silenced (shKIF5B) shRNAs were analyzed by western blotting using antibodies against kinesin-1, α-tubulin, and GAPDH. b Effects of kinesin-1-silencing on Dex-enhanced mitochondria dynamics. C2C12 cells expressing non-silenced (shCon) or Kinesin-1-silenced (shKIF5B) shRNAs were treated with the indicated concentrations of Dex for 1 h. Data are mean ± s.e.m (shCon in 0 µM Dex, n = 3481 mitochondria/6 cells; shCon in 1 µM Dex, n = 4716 mitochondria/6 cells; shKIF5B in 0 µM Dex, n = 3847 mitochondria/6 cells; shKIF5B in 1 µM Dex, n = 3578 mitochondria/6 cells). ***p < 0.001. c C2C12 cells expressing non-silenced (shControl) or Kinesin-1-silenced (shKIF5B) shRNAs were treated with the indicated concentrations of Dex for 6 h and immunostained for Tom20 (green; to visualize mitochondria), phalloidin (red; to visualize F-actin), and DAPI (blue; to visualize nucleus). Scale bar, 10 µm. d The mitochondria area (marked with Tom20) as a percentage within a cell (marked with phalloidin), as shown in c. Data are mean ± s.e.m (shCon in 0 μM Dex, n = 29 cells; shCon in 1 μM Dex, n = 30 cells; shKIF5B in 0 μM Dex, n = 30 cells; shKIF5B in 1 μM Dex, n = 30 cells). *p < 0.05; ***p < 0.001; NS no significance. e Effects of kinesin-1 silencing on Dex-enhanced myogenic differentiation. C2C12 cells expressing non-silenced (shControl) or Kinesin-1-silenced (shKIF5B) shRNAs were treated with differentiation medium containing the indicated concentrations of Dex for 3 days and analyzed by western blotting using antibodies against MYH1/2, kinesin-1, and GAPDH. f Effects of kinesin-1 on Dex-promoted myoblast fusion. C2C12 cells expressing non-silenced (shControl) or Kinesin-1-silenced (shKIF5B) shRNAs were treated with differentiation medium containing the indicated concentrations of Dex for 5 days and immunostained for MYH1/2 (yellow; myotube marker) and DAPI (to visualize nucleus). Scale bar, 100 µm. g Fusion index, calculated as the percentage of nuclei (≥3) in MYH1/2⁺ cells, as shown in f. Data are mean ± s.e.m (shControl in 0 µM Dex, n = 313 MYH1/2⁺ cells; shControl in 0.1 µM Dex, n = 236 MYH1/2⁺ cells; shControl in 1 µM Dex, n = 192 MYH1/2⁺ cells; shKIF5B in 0 µM Dex, n = 104 MYH1/2⁺ cells; shKIF5B in 0.1 µM Dex, n = 84 MYH1/2⁺ cells; and shKIF5B in 1 µM Dex, n = 79 MYH1/2⁺ cells). **p < 0.01; ***p < 0.001; NS no significance. h Effects of the association between kinesin-1 and microtubules on Dex-enhanced myogenic differentiation. C2C12 cells were treated with differentiation medium containing the indicated concentrations of Dex and kinesin-1 inhibitor RBL for 3 days and analyzed by western blotting using antibodies against MYH1/2, and GAPDH (internal control). The ratio of MYH1/2 to GAPDH is shown as fold. i C2C12 cells were treated with differentiation medium containing 40 µM RBL for 3 days and analyzed by western blotting using antibodies against MYH1/2, α-tubulin, kinesin-1, and β-actin. j Effects of the association between kinesin-1 and microtubules on Dex-promoted myoblast fusion. C2C12 cells were treated with differentiation medium containing the indicated concentrations of Dex accompanied with either DMSO (control) or 40 µM RBL for 5 days and immunostained for MYH1/2 (yellow; myotube marker) and DAPI (to visualize nucleus). Scale bar, 100 µm. k Fusion index, calculated as the percentage of nuclei (≥3) in MYH1/2⁺ cells, as shown in j. Data are mean ± s.e.m (DMSO in 0 µM Dex, n = 144 MYH1/2⁺ cells; DMSO in 0.1 µM Dex, n = 160 MYH1/2⁺ cells; DMSO in 1 µM Dex, n = 157 MYH1/2⁺ cells; RBL in 0 µM Dex, n = 130 MYH1/2⁺ cells; RBL in 0.1 µM Dex, n = 164 MYH1/2⁺ cells; RBL in 1 µM Dex, n = 34 MYH1/2⁺ cells). *p < 0.05; ***p < 0.001; NS no significance.