Fig 2: (A) Typical images under a transmission electron microscope. Red arrow indicates the normal mitochondrion, yellow arrow indicates the swollen and abnormal mitochondrion. (B) Quantification of mitochondrial number from the area of 96.9 µm2 (n = 3). (C) Quantification of mitochondrial area. (D) Levels of Cyto C, COX IV, TOM20, TIM44, and PRX3 by Western blot. (E) Quantification of (D) C, control, patients with benign disease; NC, patients of non-cachexia; CAC, cancer patients with cachexia. *p < 0.05. Data are displayed as mean ± standard deviation.

Fig 3: (A) Typical images of the C2C12 myotube cocultured with CM for 12 h. (B) Quantification of myotube diameter. (C) Levels of Atrogin-1, MuRF-1, DRP1, p-DRP1(ser616), p-DRP1(ser637), Cyto C, COX IV, and GAPDH by Western blot. (D) Quantification of Atrogin-1 and MuRF-1. (E) Quantification of Cyto C and COX IV. (F) Quantification of DRP1, p-DRP1(ser616), and p-DRP1(ser637). (G) Quantification of p-DRP1(ser616)/DRP1 and p-DRP1(ser637)/DRP1 ratio. (H) Typical images to analyze mitoROS by mitoSOX. (I) Quantification of mitoSOX with fluorescence ratio (Ex/Em = 510/580 nm). (J) Typical images to analyze MMP by JC-1. (K) Quantification of MMP with fluorescence intensity (red/green) ratio by ImageJ software. (L) Typical images to analyze mitochondrial mass by Mito-Tracker Green FM. (M) Quantification of mitochondrial mass with fluorescence intensity by ImageJ software. (N) Typical images to analyze mitochondrial morphology in myoblasts by MitoTracker Green FM. (O) Quantification of mitochondrial morphology by Mitochondrion Analyzer, a plugin of ImageJ software. FF (form factor: the reciprocal of circularity value) and AR (aspect ratio: major axis/minor axis of an ellipse equivalent to the object). (P) Quantification of MFN1, MFN2, OPA1, FIS1, and MFF; control: myotube cultured in DMEM of 2% HS; CM (conditional medium): myotube cultured in DMEM of 33% C26 conditional medium; *p < 0.05 vs. control group; #p < 0.05 vs. CM group. Data are displayed as mean ± standard deviation.

Fig 4: Effects of CIC on mitochondrial physiology of OS cells. U2OS cells were treated with melatonin, melatonin + ROCK activator and melatonin + ROCK inhibitor for 24 h. A Immunofluorescence of mitochondria in U2OS cells. Scale bar = 20 µm. B Western blot shows TOM20, COX4I1, ATP synß, PGC1a, NRF1 and TFAM expressions. C mtDNA levels of U2OS cells. D Mitochondrial ATP production in U2OS cells. E Mitochondrial O2 consumption in U2OS cells. *P < 0.05 vs. control, #P < 0.05 vs. MT. Data are expressed as mean ± SD fold induction of three independent experiments relative to control

Fig 5: Expression levels of ETC complex subunits and PGC1-NRF1/2 signaling pathway proteins during differentiation of C2C12 myoblasts. (a) Western blot of ETC complex subunits ND1, CYTB, ATP6A1, SDHA, and COXIV in C2C12 cells during differentiation on D1, D3, and D5 after treatment with UCF101 (20 µM) or no treatment and quantification of Western blots relative to GM (n = 3). (b) Western blot of PGC1a, NRF1, NRF2, and VDAC in C2C12 myoblasts at differentiation on D1, D3, and D5 after treatment with UCF101 (20 µM) or no treatment and quantification of Western blots relative to GM (n = 3). Two-tailed unpaired Student’s t-tests were used. Statistical significance: * p = 0.05, ** p = 0.01. ND1, NADH1; SDHA, succinate dehydrogenase complex flavoprotein subunit A; COXIV, cytochrome c oxidase subunit 4; CYTB, cytochrome b; ATP6, ATP synthase F0 subunit 6.