(A) Bubble plot depicting genes enriched in the forward genetic screen. Bubbles represent the genes enriched in the GFPHIGH population compared to unmutagenised KBM7 cells expressing the HIF1α-GFPODD reporter. Proteins involved in V-ATPase assembly and function (green), canonical HIF1α regulation (purple), and the oxoglutarate dehydrogenase complex (blue) are highlighted, with the number of independent gene trap insertions indicated (brackets). (B) Pathway analysis of enriched genes in the KBM7 forward genetic screen. The top 114 genes enriched for multiple independent gene-trapping integrations in the GFPHIGH population compared to unmutagenised KBM7 cells expressing the HIF1α-GFPODD reporter were analysed by gene ontology clustering for pathways significantly targeted in the screen. An individual gene enrichment p value < 0.1 was used as a threshold value for genes to be included in the pathway analysis. (C) Schematic of enriched gene trap insertion sites in the 5 V-ATPase subunits (ATP6V0D1, ATP6V1G1, ATP6AP1, ATP6V1A, ATP6V0A2) identified in the forward genetic screen. (Red = sense insertions, Blue = antisense insertions). (D, E) Validation of the V-ATPase subunits identified in the screen using CRISPR-Cas9 targeted gene editing in HIF1α-GFPODD reporter (D) and wildtype (E) HeLa cells. Cells were simultaneously transduced with Cas9 and sgRNAs to ATP6V0D1, ATP6V1G1, ATP6AP1, ATP6V1A1, or ATP6V0A2. GFP levels were assessed by flow cytometry after 10 days (% GFPHIGH cells indicated) (D), and HIF1α levels measured by immunoblot (E). PHD2 and β2m were used as positive and negative controls respectively. (F, G) Chemical perturbation of V-ATPase function. Wildtype and HIF1α-GFPODD HeLa cells were cultured in the presence of BafA (10 nM or 100 nM) for 24 hr and HIF1α levels measured by GFP fluorescence (F) or immunoblot (G).