Fig. 6: NO and HIF1α synergistically triggers IL-9 induction in Th9 cells.
From: EGFR-HIF1α signaling positively regulates the differentiation of IL-9 producing T helper cells
a–c Naïve CD4+ T cells from WT and Nos2−/− mice were differentiated to Th9 cells followed by a qPCR analysis of Spi1, Irf4, Gata3 and Batf expression. Data are representative of mean ± SEM from three independent experiments. b FACS staining for IL-9 and IL-17 in Th9 cells. c qPCR analysis of Hif1α expression. Data are representative of mean ± SEM from three independent experiments. d Bioinformatics analysis of HIF1α binding motif in Nos2 promoter followed by ChIP analysis of HIF1α binding to Nos2 promoter in Th9 cells represented as enrichment of HIF1α on Nos2 promoter relative to input. Data are representative of mean ± SEM from three independent experiments. e Luciferase assay for Nos2 promoter activity in the presence of HIF1α plasmid at 0, 50, and 100 ng concentrations. Data are representative of mean ± SEM from three independent experiments. f Naïve CD4+ T cells from WT and Hif1αkd mice were differentiated under Th9 polarizing conditions with daily treatment of 1.0 μg/ml Dox for 3 days followed by qPCR analysis of Nos2 expression. Data are representative of mean ± SEM from three independent experiments. g Naïve CD4+ T cells from WT mice were differentiated into Th9 cells under normoxic (21% oxygen) or hypoxic (1% oxygen) conditions for 3 days followed by qPCR analysis of Nos2 expression. Data are representative of mean ± SEM from three independent experiments. h Naïve CD4+ T cells from WT and Phd2kd mice were differentiated under Th9 polarizing conditions with daily treatment of 1.0 μg/ml Dox for 3 days followed by qPCR analysis of Nos2 expression. Data are representative of mean ± SEM from three independent experiments. a, c ****P < 0.00001, *P = 0.01, using two-tailed unpaired Student’s t test. d **P = 0.0017, using two-tailed unpaired Student’s t test. e ****P < 0.00001, using one-way ANOVA followed by Tukey’s multiple comparison test. f *P = 0.01, using two-tailed unpaired Student’s t test. g *P = 0.04, using two-tailed unpaired Student’s t test. h **P = 0.0026, using two-tailed unpaired Student’s t test.
