Fig 2: 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, a-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, a-tubulin, kinesin-1, and ß-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.

Fig 3: Geometric cue-coordinated cell adhesions are able to rescue the myogenic defects caused by kinesin-1 inhibition.a A polydimethylsiloxane (PDMS) stamp with micron-sized features (20 µm or 50 µm stripes) were shown in phase images. b Images of C2C12 cells plated on 20 µm or 50 µm stripe micropatterns; these were stained for fibronectin (to visualize stripe micropatterns; green), paxillin (to visualize FAs; red) and DAPI (to visualize nucleus; blue). c C2C12 cells expressing non-silenced (shControl) or kinesin-1-silenced (shKIF5B) shRNAs were plated on fibronectin-coated coverslips (with no pattern substrate), 20 µm striped coverslips or 50 µm striped coverslips, treated with differentiation medium for 5 days and immunostained for MYH1/2 (yellow; myotube marker) and DAPI (to visualize nucleus). Scale bar, 50 µm. d Aspect ratio, calculated as a ratio of the longest cell length to the shortest cell length in MYH1/2+ cells with nuclei <3 (n < 3) or nuclei =3 (n = 3), as shown in c. Data are mean ± s.e.m (shControl with no pattern with <3 nuclei, n = 68 MYH1/2+ cells; shControl on 50 µm stripes with <3 nuclei, n = 34 MYH1/2+ cells; shControl on 20 µm stripes with <3 nuclei, n = 24 MYH1/2+ cells; shKIF5B with no pattern with <3 nuclei, n = 168 MYH1/2+ cells; shKIF5B on 50 µm stripes with <3 nuclei, n = 65 MYH1/2+ cells; shKIF5B on 20 µm stripes with <3 nuclei, n = 38 MYH1/2+ cells; shControl with no pattern with =3 nuclei, n = 45 MYH1/2+ cells; shControl on 50 µm stripes with =3 nuclei, n = 52 MYH1/2+ cells; shControl on 20 µm stripes with =3 nuclei, n = 21 MYH1/2+ cells; shKIF5B with no pattern with =3 nuclei, n = 23 MYH1/2+ cells; shKIF5B on 50 µm stripes with =3 nuclei, n = 25 MYH1/2+ cells; shKIF5B on 20 µm stripes with =3 nuclei, n = 23 MYH1/2+ cells). **p < 0.01; ***p < 0.001; NS no significance. e C2C12 cells expressing non-silenced (shControl) or kinesin-1-silenced (shKIF5B) shRNAs were plated on fibronectin-coated coverslips (with no pattern substrate), 20 µm striped coverslips or 50 µm striped coverslips for 24 h and immunostained for Tom20 (green; to visualize mitochondria), a-tubulin (red; to visualize microtubules), and DAPI (to visualize nucleus). Scale bar, 50 µm. f Percentage of mitochondria area (marked with Tom20) within a cell (marked with a-tubulin), as shown in e. Data are mean ± s.e.m (shControl with no pattern, n = 32 cells; shControl on 50 µm stripes, n = 22 cells; shControl on 20 µm stripes, n = 20 cells; shKIF5B with no pattern, n = 39 cells; shKIF5B on 50 µm stripes, n = 34 cells; shKIF5B on 20 µm stripes, n = 20 cells). **p < 0.01; ***p < 0.001. g Fusion index, calculated as the percentage of nuclei (=3) in MYH1/2+ cells, as shown in c. Data are mean ± s.e.m (shControl with no pattern, n = 154 MYH1/2+ cells; shControl on 50 µm stripes, n = 112 MYH1/2+ cells; shControl on 20 µm stripes, n = 53 MYH1/2+ cells; shKIF5B with no pattern, n = 165 MYH1/2+ cells; shKIF5B on 50 µm stripes, n = 108 MYH1/2+ cells; shKIF5B on 20 µm stripes, n = 56 MYH1/2+ cells). *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 4: GDAP1 silencing increases mitochondrial proteins for Complex V and II. Primary human skeletal muscle cells were used for western blot analysis of protein abundance. Cells were transfected with a non-targeting scrambled control RNA (open squares) or siRNA targeted against GDAP1 (black circles) and treated with regular media (Unt), AICAR (AIC), or phenformin (Phen). (A) p-ACC2S222. (B) ACC2. (C) MYH1/2. (D) Desmin. (E) OXPHOS Complex V. (F) OXPHOS Complex III. (G) OXPHOS Complex II. (H) OXPHOS Complex IV. (I) OXPHOS Complex I. (J) ß-Actin. All data are plotted from n = 7–8 matched samples. *Significant pairwise difference between subsets of data indicated by brackets as detected by pairwise post hoc tests after FDR-correction, †Significant differences among groups as detected by Friedman's test (assumptions for 2-way-RM ANOVA were not met), ‡Significant effect of pharmacological treatment as detected by 2-way-RM ANOVA, §Significant effect of GDAP1 silencing as detected by 2-way-RM ANOVA.