Cells were infected with a pLX304-EV-IRES-GFP (EV-GFP), pLX304-VHL-IRES-Tdtomato (VHL-Tdtomato), or pLX304-VHLΔB-IRES-Tdtomato (VHLΔB-Tdtomato) lentivirus as indicated, followed by selection for antibiotic resistance with blasticidin (10 μg/ml). For competition assays with small-molecule inhibitors, EV-GFP and VHL-Tdtomato (or VHLΔB-Tdtomato) were mixed (1:1) and seeded at 300,000 cells/10-cm dish and treated with the indicated concentrations of drug or the equivalent volume of vehicle. The cells were split every 3 to 4 days. After each split, a portion of the cells were reseeded in fresh media and drug, and the remaining cells were used for flow cytometry analysis.

For competition assays using CRISPR/Cas9 editing of target genes, EV-GFP and VHL-Tdtomato cells were mixed (1:1) and seeded at 300,000 cells per well in a six-well dish and allowed to attach for at least 6 hours. The cells were then infected with viruses encoding sgRNAs against the desired target as described above. After each split, a portion of the cells were reseeded in fresh media and drug, and the remaining cells were used for flow cytometry analysis.

For flow cytometry, 10,000 cells per sample were analyzed using a BD LSRFortessa flow cytometer with the BD FACSDiva software. Living single cells were gated, and then the percentages of those cells that were GFP positive or Td-tomato positive were quantified. The ratio of Tdtomato-positive:GFP-positive cells was used as a measure of VHL+/+:VHL−/− cells and normalized to the ratio in the vehicle-treated or nontargeting sgRNA sample for each time point.

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