Glucose uptake assay

Glucose uptake activity of the cells after the treatment of E6155 with or without EX527 was assayed by 2-NBDG as described previously [15]. L02 or L6 myotubes were incubated with E6155 (0.1 μM) with or without EX527 (10 μM) in 37 °C for 18 h. After that, cells were stimulated with 100 nM insulin for 30 min. Then, insulin was replaced with 50 μM of 2-NBDG. After 1h, the cells were washed three times and lysed in lysis buffer for 10 min. The fluorescence intensity of supernatants which containing equal amount of protein were measured using a fluorometric plate reader (PerkinElmer) at 485 nm excitation and 535 nm emission, respectively. Each glucose uptake assay was performed three times.