2 deoxy d glucose hy 13966

The following antibodies were used in this study: rabbit anti-Myo19 (HPA059715, 1:1000 for western blotting) from Sigma-Aldrich; rabbit anti-Mic60 (A2751, 1:3000 for western blotting), anti-Metaxin2 (A7958, 1:1000 for western blotting) and rabbit anti-Sam50 (A3401, 1:3000 for western blotting, 1:400 for PLA assay) from ABclonal; mouse anti-Mic60 (sc-390707, 1:200 for immunofluorescence staining) from Santa Cruz Biotechnology; rabbit anti-GFP (598, 1:1000 for western blotting, 1:200 for immunofluorescence staining) and mouse anti-GFP (MO48-3, 1:200 for immunofluorescence staining) from MBL; anti-mouse (sc-516102, 1:4000) and anti-rabbit (sc-2004, 1:4000) horseradish peroxidase (HRP)-conjugated secondary antibodies from Santa Cruz Biotechnology; Alexa Fluor488- or Alexa Fluor 555-conjugated secondary antibody from ThermoFisher Scientific. For reagents, MitoTracker^TM Red CMXRos (M7512), MitoTracker^TM Green FM (M7514), TMRE (T669), TMRM (T668) and Prolong Diamond Antifade with DAPI (P36962) were purchased from Thermo Fisher Scientific. Rotenone (HY-B1756), FCCP (HY-100410) and 2-Deoxy-D-glucose (HY-13966) were purchased from MedChemExpress.

Cells were seeded in 35 mm glass-bottom dishes twelve hours before transfection with the plasmid. Twenty-four hours after transfection, Hoechst 33342 (4082, Cell Signaling Technology, CST, Danvers, MA, USA) was added to the medium at a final concentration of 1 μg/mL, and the cells were incubated for 10 min at 37 °C. Hoechst-containing medium was replaced with fresh complete medium before the cells underwent live-cell imaging using a Zeiss LSM880 confocal microscope equipped with an incubation chamber to provide a humidified atmosphere at 37 °C with 5% CO₂. 1,6-Hexanediol treatment: Cells cultured on 35 mm glass-bottom dishes with complete medium were imaged every 2 s. After the 10th acquisition, cells were incubated with complete medium containing 10% 1,6-hexanediol. After the cells were incubated for 60 s, the culture medium was replaced with complete medium, and the cells were cultured for another 60 s. ATP depletion: Complete DMEM was replaced with glucose-free DMEM (11966025, Gibco), and the cells were cultured for 2 h. 2-Deoxy-D-glucose (HY-13966, MedChemExpress, MCE, Monmouth Junction, NJ, USA) and oligomycin (495455, Sigma-Aldrich, St. Louis, Missouri, USA) was added to the medium at final concentrations of 5 mM and 126 nM, respectively, after which the cells were cultured for another two hours before observation.