Fig. 5

Generation of CRISPR/Cas9-mediated knockout K562 lines for HIF1, HIF2, and ARNT. a Single-cell-derived knockout lines were generated and validated by sequencing after which 4 single cell-derived lines were pooled for further analyses. Cells were grown for 24 h under hypoxia and extracts were western blotted for the presence of HIF1, HIF2, and ARNT. b K562 cells were treated as in a, and ChIP Q-PCR experiments were performed. The knockout is shown below the x-axis, the antibodies used for ChIP-PCRs are shown in the colored boxes (HIF1 in top panels, HIF2 in middle panels, ARNT in lower panels), and the loci to which binding is investigated is shown at the top (ALDOA for left panels and GPI for right panels). c Q-PCR was performed on knockout lines. Cells were grown under hypoxia for 24 h. d Experiment as in c but now cells were grown under hypoxia for 10 days to evaluate gene expression changes under chronic hypoxia conditions