Extended Data Fig. 1: Genome-wide mutagenesis screen identifies genes required to activate a HIF response.
a-g, Screen validation using CRISPR-Cas9 targeted depletion in mixed KO populations. HeLa HRE-ODDGFP reporter cells were transduced with sgRNAs against a, HIF1β or UBE2A; b–d, β2m; e, RNF20 or RNF40; f, HIF1β or PPP4C; g, SET1B. Cells were treated with 21% or 1% O2 for 24 h or DMOG 1 mM for 24 h, and GFP levels measured by flow cytometry. h, i, KO clones of HIF1β and SET1B were generated in HeLa HRE-GFPODD reporter cells by serial dilution and validated by immunoblot (representative of 3 experiments) (i). h, Control or KO clones were incubated at 21% or 1% O2 for 24 h and the GFP levels were measured by flow cytometry (n=3 biologically independent samples). j, Loss of SET1B or HIF1β does not alter basal reporter activity. HIF1β and SET1B mixed KO populations of HeLa HRE-ODDGFP reporter cells were generated using CRISPR-Cas9 targeted depletion, and GFP levels were measured with flow cytometry.
