2-NBDG uptake

A total of 2 × 10^5 cells were seeded in 6-well plates and incubated overnight. The cells were treated as indicated for 24 h. After treatment, to measure glucose uptake, the cells were washed with PBS twice. Then, they were incubated in glucose-free Dulbecco’s Modified Eagle Medium (DMEM) containing 1% BSA, and 1 mM 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG; Thermo Fisher Scientific) for 30 min at 37 °C in a cell incubator. Cells were washed quickly and thoroughly three times with 2 mL of ice-cold phosphate-buffered saline (PBS).  Ensure complete removal of extracellular 2-NBDG. The cells were analyzed by fluorescence microscopy (Olympus, Tokyo, Japan).