Cell tak

Glycolysis and glucose uptake assays using ³H-glucose or ³H-2-deoxyglucose have been described previously (Gerriets et al., 2015 (link); Macintyre et al., 2014 (link); Wang et al., 2011 (link)). Pentose phosphate pathway (PPP) flux and glucose oxidation were determined by the rate of ¹⁴CO₂ release from 1-¹⁴C-glucose as described (Wang et al., 2011 (link)). All values were normalized to cell number. OCR and ECAR were measured with an XF24 extracellular flux analyzer (Seahorse Bioscience) as described (Caro-Maldonado et al., 2014 (link)). Suspension cells were attached to culture plates using Cell-Tak (BD Bioscience). OCR and ECAR were measured in unbuffered DMEM (Sigma-Aldrich) supplemented with 10mM D-glucose (Sigma-Aldrich) and 10mM L-glutamine, as indicated. OCR and ECAR values were normalized to cell number. For certain experiments, OCR was measured over time following injection of 1 μM oligomycin.

OCI-AML3 cells were treated with 0.1% DMSO; 1 μM CB-839; 0.5, 1, 2, or 4 mM DMKG; or 1 μM CB-839 + 0.5, 1, 2, or 4 mM of DKMG for 6, 12, or 24 hours in RPMI medium containing 10% fetal bovine serum. Cells were washed with phosphate-buffered saline solution; resuspended in XF Assay medium supplemented with fresh glucose 10 mM, glutamine 2 mM, and pyruvate 1 mM; plated in XF96e Seahorse Biosciences plates pre-coated with Cell-Tak (BD Biosciences, San Jose, CA) at a density of 3 × 10⁵ cells per well; and spun down on the plate to allow cells to adhere equally. Six replicate wells were analyzed for each treatment.

Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was measured using the Seahorse XF^e24 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA) according to manufacturer’s instructions and further detailed by Wu et al. (Wu et al., 2007 (link)). Briefly, 1×10⁶ TCL1/p53 cells isolated from splenocytes were seeded in a 24-well cell culture plate (100777-0044, Seahorse Bioscience) coated with Cell-Tak (354240, BD Bioscience, Bedford, MA) in DMEM supplemented with 2g/L glucose, 1mM sodium pyruvate and 2mM glutamine, titrated to pH 7.4 with sodium hydroxide. Plates were centrifuged on a swing-bucket rotor for 5 min at 150x g (accel 1, brake 1) followed by rotating plate and repeating centrifugation for 5 min at 150x g (accel 1, brake 0). Cells were then incubated in a CO₂ free incubator 1 h before the assay. OCR and ECAR was measured every 8 min followed by sequential injection of assay media, oligomycin, FCCP and rotenone up to 2.5h. Rates for OCR and ECAR were normalized to the cell number in each well and presented as relative units: OCR (pmol/min/1×10⁶ cells) and ECAR (mpH/min/1×10⁶ cells).

Utricles harvested from P3-P5 Lgr5^{EGFP-CreERT2/+}; Rosa26R^tdTomato/+ mice were attached to 35 mm glass bottom dishes (MatTek) pre-coated with CellTaK (BD Biosciences). Whole organs were cultured overnight, then treated with neomycin (1.0 mM × 24hr, Sigma) as above. 4OH-tamoxifen (500 nM, Sigma) in growth factor-enriched, serum-free media was present for 2–4 days first. Then organs were imaged in DMEF/F12 without phenol red (1:1 Cellgro) media using a spinning disc confocal imaging system (Zeiss Axio Observer Z1 or OlympusIX-81 coupled with a Yokogawa spinning disc system CSU-X1A 5000) connected to an incubating chamber (37°C, 5% CO₂). One to two EGFP+ regions with tdTomato+ cells were selected from each utricle, and z-stack images spanning the sensory epithelium were taken at 0.5–1 hr intervals. Collected videos were processed and analyzed with ImageJ64 (NIH), MetaMorph (NX 2.0; Olympus) and Volocity software (v6.1.0; Improvision).