Fig 5. Oscillating hypoxic response can lead to differential gene regulation and allow proliferation under hypoxia.

(A) Experimental protocol to entrain intracellular hypoxic response by imposing oscillations of extracellular O₂. (B) Immunoblot showing HIF-1α abundance in HeLa cells after 1% hypoxia for 6 hours, and after re-oxygenation for 10 min, and 1 hour, GAPDH is loading control; (C) RNA-Seq based hierarchical clustering of gene expression in HeLa cells kept in normoxia, and stable or oscillating hypoxia for > 24 h; Genes differentially regulated by oscillatory hypoxia shown in bracket. (See Fig S5). (D) Key Ingenuity pathways upregulated in gene-set differentially regulated by oscillatory hypoxia (referred to in B) vs persistent hypoxia. (E-J) Oscillations in HIF-1α abundance allows cells to continue to proliferate in hypoxia; (E) Immunoblot showing abundance of Cyclin B1 and Cyclin D1 in cells in normoxia, and stable or oscillating hypoxia; GAPDH is loading control. (F) RNA-Seq-based analysis of pairwise fold-changes between cells in above conditions for cell-cycle related genes. (G) Propidium iodide levels in HEK293 cells after conditioning with stably high, low or oscillatory input of 2% oxygen for 18 h; Quantification of gated cycling cells shown in (H); n = 12 exp. (I) Percentage of HypoxCR-GFP transfected HeLa cells positive for Geminin-mCherry after 12 h each of environmental normoxia, hypoxia, and oscillating hypoxia; above panel shows a representative location. (J) Percentage of HypoxCR-GFP transfected HEK293 cells positive for Geminin-mCherry within HRE-GFP+ve, HRE-GFP-ve subpopulations, and cells that spontaneously oscillate in persistent hypoxia; n = 6 exp. (K) Percentage of proliferating cells in HeLa cells stably transfected with Tet-inducible wild-type HIF-1α (HIF-1α^WT) and HIF-1α with mutation in KFERQ-like lysosomal targeting motif (HIF-1α^ΔΔ), with stable or oscillating doxycycline concentrations; n = 6 exp. Error bars: s.e.m.; *: p < 0.05, **: p < 0.01, ***: p < 0.001.