Glucose consumption and Oil Red O staining
Initially, the cardiomyocytes and differentiated skeletal myoblasts were seeded in serum-free and low-glucose DMEM medium for 24 h starvation. The cells were treated with PA and/or samples for another 24 h period. The supernatant of each group was collected at 8, 16, 24, and 48 h for analysis. Glucose consumption in the supernatant was measured with a commercial kit.
Lipid droplets within cardiomyocytes and differentiated skeletal myoblasts were observed using Oil Red O staining. Briefly, the cells were washed with phosphate-buffered saline (PBS) and fixed with 4% formaldehyde for 20 min. The formaldehyde was substituted in PBS. Subsequently, the cells were washed with 60% isopropanol and kept for 5 min. Lipid droplets in cells were stained by Oil Red O for 30 min incubation, followed by two times of PBS washing. PBS was discarded, and cells were mounted with hematoxylin solution for nuclear staining. The stained cells were washed and imaged by a microscope (Olympus CKX53, Tokyo, Japan).