Fig 1: Palmitate-induced lipotoxicity in C2C12 muscle cells. C2C12 muscle cells were treated either with control shRNA (Con shRNA) or Cs shRNA which targeted Cs mRNA. Afterwards Con shRNA and Cs shRNA cells were incubated in the media containing 5.5 mM glucose (G) and/or 0.8 mM palmitate (P). (a) Cell proliferation was assessed using cell counting assay kit-8 (96992, Sigma, UK) and verified by (b) crystal violet staining; (c) cellular ATP concentrations were assessed using ATP calorimetric assay (ab83355, Abcam, Cambridge, UK); (d) levels of cleaved caspase-3 were assessed using immunoblotting; (e) production of reactive oxygen species was assessed by measuring 2', 7'-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescence (values are means ± SEM). **P < 0.01, B6 vs B6.A strains. †p < 0.001 G vs G + P; ***P < 0.001, Con shRNA vs Cs shRNA.
Fig 2: CCCP treatment causes an imbalance in mitochondrial dynamics in C2C12 myotubes. A, The mitochondrial membrane potential was evaluated as described in the Materials and Methods (left). Intracellular ATP levels were measured in C2C12 myotubes treated with 10 µmol/L CCCP in the presence or absence of 2 mmol/L NAC (right). ATP levels were estimated by using an ATP colorimetric/fluorometric assay kit (Abcam, ab83355) according to the manufacturer's instructions (B) Changes in the mRNA levels of mitochondrial OXPHOS complex subunits after CCCP treatment (C) Measurement of ROS in C2C12 myotubes as described in the Materials and Methods. Scale bars = 50 µm. D, Changes in the expression of proteins related to mitochondrial fusion and fission. E, Immunoblot analyses of proteins related to mitochondrial dynamics in C2C12 myotubes. All results are representative of more than three independent experiments. The data represent the mean ± SEM. P value < .05 obtained by one-way ANOVA was considered statistically significant
Supplier Page from Abcam for ATP Assay Kit (Colorimetric/Fluorometric)