Fig. 5: Chkb deficiency results in decreased fatty acid usage and increased lipid droplet accumulation in differentiated myocytes in culture.

A Representative image of isolated skeletal myoblasts from Chkb^+/+ and Chkb^−/− mice, cultured on Matrigel® coated culture flasks. At day 0, when the cells reached 80% confluency, the medium was replaced by differentiation medium and maintained in differentiation media for up to 8 days. B Western blot of differentiated Chkb^+/+ and Chkb^−/− myocytes probed with anti-Chka, anti-Chkb, and anti-Gapdh antibodies. For A and B, Each experiment was repeated independently 3 times with similar results. C RT-qPCR analysis of gene expression in isolated myocytes from Chkb^+/+ and Chkb^−/− mice at day 5 of differentiation. Values are means ± SEM; n = 3 independent experiments. Two-sided student’s t-test, p = 0.0200 (Chka), p = 0.0095 (Icam1), p = 0.01944 (Tgfb1). *p < 0.05, **p < 0.01. D Formaldehyde fixed and immunostained myotubes were categorized into three groups (1 to 3 nuclei, 4 to 10 nuclei, and >10 nuclei per myotube). The number of multinuclear myotubes in the two groups and the distribution of nuclei were calculated to compare differentiation in primary Chkb^+/+ and Chkb^−/− myocytes. For D values are means ± SEM. n = 5 random images from 3 independent experiments for each group. Two-sided student’s t-test did not result in p values less than 0.05. Representative traces of oxygen consumption rates (OCRs) of primary Chkb^+/+ and Chkb^−/− myocytes at day 4 (E, F) and day 8 (G, H) of differentiation driven with glucose/glutamine/pyruvate or palmitate as stated. Bovine serum albumin (BSA) alone was used as control for palmitate-BSA complex driven OCRs. Oligomycin(O), FCCP(F), rotenone (R) and antimycin A (AA) were sequentially injected to assess mitochondrial respiratory states. Data are mean ± SD. For E, n = 5 (Chkb^+/+) and n = 5 (Chkb^−/−) wells per group. For (F), n = 5 (BSA Chkb^+/+), n = 5 (BSA Chkb^−/−), n = 5 (Palmitate-BSA Chkb^+/+), n = 5 (Palmitate-BSA Chkb^−/−). For G, n = 10 (Chkb^+/+) and n = 8 (Chkb^−/−) wells per group. For (H), n = 4 (BSA Chkb^+/+), n = 4 (BSA Chkb^−/−), n = 4 (Palmitate-BSA Chkb^+/+), n = 4 (Palmitate-BSA Chkb^−/−). For E–H Two-sided student’s t-test. For E, during maximal respiration, p = 0.0062, p = 0.0021, p = 0.00187. For F, during maximal respiration, p = 0.0857, p = 0.0421, p = 0.0526. For G, during maximal respiration, p = 0.1242, p = 0.1139, p = 0.0713. For H, during maximal respiration, p = 0.01231, p = 0.0072, p = 0.0038. *p < 0.05. I Isolated primary myocytes from Chkb^+/+ and Chkb^−/− mice were fixed 5 days after differentiation and stained with BODIPY-493/503 to visualize LDs (green). DAPI was used to stain nucleus (blue). J The corrected total cell fluorescence intensity of lipid droplets was significantly enhanced in Chkb^−/− myotubes. Box plots in J show median, quartiles (boxes), and range (whiskers). For J, n = 16 (Chkb^+/+) and n = 15 (Chkb^−/−) myotubes. For J Two-sided student’s t-test. For J, p = 0.0463. *p < 0.05. I and J are representative of 3 independent experiments with similar results. Source data are provided as a Source Data file.