Fig 2: Estrogen controls HIF-1α stabilization via the HSP70-mediated degradation pathway. a Volcano plot of RNA-seq analysis of estrogen (E2) (1 μmol·L−1)-treated versus vehicle (Veh)-treated WT bone marrow B cells under hypoxic conditions showing genes upregulated and downregulated, respectively, with a fold change (FC) higher than 2. b Heatmap of differentially expressed genes found within gene sets displayed in (a). c Hspa1a, Hsp90aa1, Hsp90ab1 and Hif1a mRNA expression in B cells treated with the indicated concentration of estrogen (E2) under hypoxia for 12 h (n = 3). Values for the control group were set as 1. d Hif1a, Vhl, Phd1, Phd2, Phd3, Hspa1a, Hsp90aa1, and Hsp90ab1 mRNA expression in B cells from the bone marrow of sham control (Sham) and ovariectomized (OVX) mice. e Levels of VHL, PHD1, PHD2, PHD3, HSP70, and HSP90 protein in isolated B cells from the bone marrow of OVX and Sham control mice (n = 3). f HIF-1α, HSP70, HSP90, hydroxylated HIF-1α (P564 and P402 sites), PHD1, PHD2, PHD3, VHL and β-actin expression in isolated bone marrow B cells with the indicated concentration of estrogen (E2) treatment under hypoxic culture for 24 h. g HIF-1α, HSP70 and β-actin expression in bone marrow B cells transfected with control or HSP70 siRNA lentivirus under hypoxic culture for 24 h. MG132 (100 μmol·L−1) was added to HIF-1α immunoprecipitation samples. *P < 0.05, **P < 0.01, ***P < 0.001

Fig 3: HIF-2α-KO cells have high expression of HIF-1α.a CD45.2+ tTreg cells were transferred to CD45.1+ RAG-1-KO mice together with CD45.1+ Teff. CD45.2+ tTreg cells were re-isolated after 15 days, activated by TPA/A23187 (50/500 ng ml−1), and the intracellular HIF-1α, Foxp3, and IL-17 determined. n = 3 (Hif2af/f), 4 (Cd4CreHif2af/f). Data represent mean ± SEM. *P = 0.011 (IL-17A+), 0.0422 (HIF-1α+) by two-tailed unpaired t-test. b, c Hif1a in Hif2af/f and Cd4CreHif2af/f naïve CD4+ T (b) and tTreg (c) cells was determined by quantitative PCR. n = 3. *P = 0.0116 b, ***P < 0.001 c by two-tailed paired t-test. d WT and HIF-2α-KO CD4+ naïve T cells were activated by CD3/CD28 (2/1 μg ml−1) and HIF-1α levels were determined. e, f Hif2af/f and Cd4CreHif2af/f tTreg cells were activated by CD3/CD28 (4/2 μg ml−1) for 24 h under normoxic or hypoxic conditions (e), or iTreg cells were activated by CD3/CD28 (2/1 μg ml−1) for 4 h (f), and HIF-1α levels were determined. Each sample f represents independent iTreg cells preparation. g Hif2af/f and Cd4CreHif2af/f iTreg cells were activated as in f under hypoxia for 4 h. Cells were then treated with cycloheximide (100 μg ml−1) and HIF-1α levels determined. h, i WT naïve CD4+ T cells (h) were activated by CD3/CD28 (2/1 μg ml−1) in the presence of HIF-2α inhibitor PT2385 for 2 days, or activating WT iTreg cells (i) were treated with 10 μM PT2385, and HIF-1α levels determined. Results have been reproduced in three (d, h) or two e–i independent experiments. j WT tTreg cells were stimulated with or without 10 μM PT2385, and IL-17 production was quantitated. Data are technical triplicates, the experiment was independently reproduced twice. k Hif2af/f and Cd4CreHif2af/f tTreg cells were analyzed for expression of Glut1 and Ccr4 by quantitative PCR. n = 4 (Glut1), n = 5 (Ccr4). *P = 0.0236 (Glut1) and 0.0488 (Ccr4), by two-tailed paired t-test. l The expression of Hif2a in Hif1af/f and Cd4CreHif1af/f naïve CD4+ T and tTreg cells was determined. n = 3. *P = 0.0464, ***P < 0.001, by two-tailed paired t-test. Source data are provided as a Source data file.

Fig 4: The hypoxia-induced G1-arrest causes continued radioresistance after reoxygenation. (A) Typical cell cycle distribution of RPE cells grown for 72 h in normoxic or hypoxic conditions (1% O2). The difference of each cell cycle phase fraction between hypoxic and normoxic cell cycle distribution (Δcell cycle population fraction) is depicted. (B) Western blot analysis of cells that have been cultured in normoxia (OO) or hypoxia (HO) before being irradiated in aerated conditions. Western blot samples were collected after culturing cells for 72 h in normoxia and hypoxia, at the time of irradiation in aerated conditions and 30 min post-IR. HIF1a as a marker of active hypoxic signaling, Cyclin E1 as a marker of quiescence, and pRB (807/811) as a marker of active transition from G1 to S phase are shown (C) Graphical representation of the FUCCI system. Typical cell density plots acquired from mock-irradiated RPE FUCCI cultivated either under normoxic or hypoxic conditions. The cell cycle profile based on the expression of the red and green fluorescence and the sorting of the populations is depicted (see material and methods text for more details). (D) Graphical representation of the experimental design. (E) The Surviving fraction of different cell subpopulations (indicated in B) after 2 and 4 Gy is shown. The bars represent the mean differences of three independent experiments, and the error bars the 95% C.I. of the means.

Fig 5: Percent tissue HIF1-α positive in SCCVII tumors. Error bars depict ±SD, N = 6 for each group (p < 0.05 between control and 12 h)