Fig. 3

IRE1α/XBP1 and HIF1α crosstalk regulates the myeloid inflammatory response secondary to a hypoxic stimulus. A Co-immunoprecipitation of XBP1 and immunoblot for HIF1α in cytosolic and B nuclear fractions of hypoxic (2% O₂ for 1 h) J774 cells (n = 2 independent experiments). C, D Schematic representation and RT-qPCR analysis of Il6, Il1b, and Vegfa mRNA expression in hypoxic (2% O₂ for 8 h) J774 cells preincubated for 1 h with IRE1α endoribonuclease inhibitor 4µ8c (100µM) or IRE1α kinase inhibitor KIRA6 (1µM) (n = 3–8 per condition). E HIF1α or mock (IgG) ChIP-qPCR at Il6, Il1b, and Vegfa loci in hypoxic (2% O₂ for 8 h) J774 macrophages preincubated for 1 h with IRE1α endoribonuclease inhibitor 4µ8c (100µM) or IRE1α kinase inhibitor KIRA6 (1µM) (n = 3 independent experiments). Percent of input represents the signals obtained from the HIF1α ChIP over signals from respective input samples. Data expressed as mean ± S.E.M. Statistical analysis (D, E): one-way ANOVA with Bonferroni post hoc analysis; *P < 0.05, **P < 0.01, and ***P < 0.001