2-NBDG glucose uptake assay

HepG2 cells were seeded in six-well plates (8 × 10⁵ cells/well) in DMEM/F-12 GlutaMAXTM (Gibco) supplemented with 10% of FBS and 1% antibiotics (Penicillin/Streptomycin). Cells were treated with 100 μΜ of C1632 for 4 days. On the day of the flow cytometry assay cells were incubated in glucose-free DMEM medium (Gibco) at 37 °C for 2 h. Cells were then incubated with 200μΜ of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]−2-deoxy-D-glucose (2-NBDG) (Biovision) at 37 °C for 45 min. Cells were washed with phosphate-buffered saline (PBS) and subsequently detached using trypsin for flow cytometry analysis. Cells were washed with 1 ml PBS prior to resuspension in 200 µl FACS buffer (2% FBS in PBS) and subsequently strained through CellTrics 50μm filters. For each measurement, data from 20 × 10³ single viable cell events were acquired using a Flow cytometer (BD LSR Fortessa) using the blue laser (excitation: 488 nm) with emission at 530/30 nm (FL-2 photomultiplier). The gating strategy was designed to exclude dead cells and cell doublets. Data were analysed with FlowJo software (BD Biosciences).