Cellular OCR was measured using XF96 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA). Briefly, HL-60 cells were seeded in XF96-well plates (20,000 cells per well in 80 μl). After 1 hour, cells were incubated overnight at 37°C, 5% CO2. The XF96 sensor cartridge was hydrated with 200 μl of calibration buffer per well overnight at 37°C. Cells were preincubated with or without IR-34 (5 μM) for 1 hour before the bioenergetic profile was determined. Then, cells were washed twice with 200 μl of prewarmed base medium containing 10 mM sodium pyruvate and 25 mM glucose. The sensor cartridge was loaded with assay media (ports A, B, and C) to measure basal OCR or with oligomycin (2.0 μM, port A), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (2.0 μM, port B), and rotenone (0.5 μM, port C) to measure the bioenergetic profiles of different cells.

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