Sc1722

For Western blots, ALL cells were infected with individual shRNAs coexpressing GFP to a final infection percentage of 40%–60% and sorted based on GFP expression, and total protein was isolated from this sorted cell population. Proteins were detected with the following antibodies: Phf6 (1:1000 in TBS-T with 5% BSA; Novus Biologicals, NB100-68262), Runx1 (1:1000 in TBS-T with 5% BSA; Abcam, ab23980), and HSP60 (1:1000 in TBS-T with 5% BSA; Santa Cruz Biotechnology, SC1722). ImageJ was used to perform protein densitometry.

Cells from the extracellular lactate experiments were lysed in RIPA buffer containing 5% protease inhibitors and 1% phosphatase inhibitors, and the protein concentration was quantified as indicated above. The samples were then denatured in Laemmli buffer, resolved on SDS-polyacrylamide gels and transferred to 0.45 μm pore size nitrocellulose membranes (10600007, Cytiva). The membranes were then blocked and the human cell lines were probed with antibodies against HIF1α (610959, BD Transduction Laboratories), CAIX (sc-365900, Santa Cruz Biotechnology), GAPDH (ab9485, abcam), GOT1 (14886-1-AP, Proteintech), GOT2 (HPA018139, Sigma-Aldrich), HSP60 (ab82520, abcam) and β-actin (A3854, Sigma-Aldrich), while the mouse cells were probed with antibodies against HIF1α (10006421, Cayman Chemical), PDK1 (ADI-KAP-PK112-F, Enzo Life Sciences, Inc.) and HSP60 (sc-1722, Santa Cruz Biotechnology). Antibody binding was detected with the Clarity Western ECL Substrate (1705061, Bio-Rad) or Super-Signal West Femto Maximum Sensitivity Substrate (34096, Thermo Fisher Scientific), and visualized on a digital luminescent image analyzer (Image Quant LAS4000 Mini, GE Healthcare).