Fig. 3.

Oxygen chemotaxis is independent of the PHD/HIF pathway. a–d HIF1A, HIF2A, HIF1A+HIF2A and PHD2 CRISPR/Cas9 KO clones characterisation, respectively, regarding O₂-directed migration. Left panels: immunoblot validation of HIF1A, HIF2A, HIF1A+HIF2A and PHD2 KO clones. To blot HIFs factors (a, b, c), cells were first pre-treated for 5 h with CoCl₂ 300 µM before protein extraction, a condition that promotes HIF factors accumulation (cf. Supplementary Fig. 8f). Middle panels: cell trajectories under confinement. Red dashed lines indicate the border of the cell cluster at 0 h. Right panels: relative distribution of MCF10A KO clones versus wt cells at the edge of the cluster at 48 h. These experiments demonstrate that HIF factors deletion does not prevent aerotaxis. e, f Tracks and redistribution of wt and PHD2 KO clone silenced for PHD3 (siPHD3) or not (siCTR). g Tracks and redistribution of MCF10A cells treated with DMOG (50 µM) or CoCl₂ (50 µM) to inhibit PHDs, or with vehicle only (DMSO). These experiments demonstrate that PHDs do not participate in O₂-sensing during aerotaxis. Confinement was applied for 48 h (a–g). Scale bars, 500 µm