Fig. 2
From: HIF1α-dependent hypoxia response in myeloid cells requires IRE1α
IRE1α kinase activity is required for HIF1α stabilization in macrophage response to hypoxia. A Immunoblot timecourse from J774 macrophage cell lysates under hypoxia probed for HIF1α stabilization, IRE1α phosphorylation and expression. (n = 3 independent experiments). B Co-immunoprecipitation of HIF1α and IRE1α in hypoxic (2% O2 for 1 h) J774 macrophages preincubated for 1 h with IRE1α endoribonuclease inhibitor 4µ8c (100µM) or IRE1α kinase inhibitor KIRA6 (1µM) (n = 3 independent experiments). Red box highlights Co-IP results upon KIRA6 treatment. C Immunoblots for HIF1α stabilization in cytosolic and nuclear fractions of hypoxic (2% O2 for 1 h) J774 cells pretreated with IRE1α kinase inhibitor KIRA6 (1µM) for 1 h. LDH was used to assess the purity of the cytosolic fraction (n = 3 independent experiments). D RT-qPCR analysis of Hif1a mRNA expression in hypoxic (2% O2 for 8 h) J774 cells preincubated for 1 h with IRE1α kinase inhibitor KIRA6 (1µM). n = 3–8 per condition, unpaired two-tailed t-test. E RT-qPCR analysis of Hif1a and F Ern1 mRNA expression in LysM-Hif1a−/− or LysM-Ern1−/− peritoneal macrophages and their control LysM-cre/HIF1a+/+/Ern1+/+ mice under normoxic (21% O2) or hypoxic (2% O2 for 8 h) conditions. n = 3–12 per condition. Data expressed as mean ± S.E.M. Statistical analysis (D, F, G): one-way ANOVA with Bonferroni post hoc analysis; *P < 0.05, **P < 0.01, and ***P < 0.001