Fig 2: During acute hindlimb ischemia, muscle tissue expression of pro-angiogenic VEGF-A is dependent on macrophage IL-1ß despite intact activation of hypoxia-driven transcriptional mechanisms via HIF-1a(A–C) Representative VEGF-A and IL-1ß immune-blots from ischemic muscle tissue at day 3 post femoral artery ligation from control or myeloid IL-1ß-deleted mice (mIL-1ß KO) with each lane containing muscle lysate from a separate animal along with quantification (B, C) of ß-actin-normalized protein (***, p < 0.0001 by t test; n = 6 mice, three males and three females).(D) Quantitative RT-PCR for relative VEGF-A165a mRNA expression from ischemic muscle of animals treated as in (A) (***, p < 0.0001 by t test; n = 6 mice total, three males and three females).(E) Quantitative RT-PCR for relative VEGF-A165b mRNA expression from ischemic muscle of animals treated as in (A) (***, p < 0.0001; n = 6 mice total, three males and three females).(F) Modified ELISA for relative HIF-1a activity on nuclear fraction lysates from day 3 ischemic muscle tissue of animals treated as in (A) (n = 6 mice total, three males and three females).(G) BMDMs from control or mIL-1ß KO mice were treated with either LPS+IFN-? or IL-4+IL-13 in the setting of normoxia or hypoxia (1% O2) followed by modified ELISA for relative HIF-1a activity on nuclear fraction lysates (***, p = 0.0001 compared to normoxia control treated with LPS+IFN-? by ANOVA; n = 4 mice total, two males and two females).(H) BMDMs from control or mIL-1ß KO mice were treated with LPS+IFN-? in the setting of normoxia or hypoxia (1% O2) followed by ELISA on culture supernatants for secreted VEGF-A protein (***, p = 0.0001 by ANOVA; n = 4 mice total, two males and two females).(I) BMDMs treated as in (H) followed by quantitative RT-PCR for relative VEGF-A165a mRNA expression (*, p < 0.05; ***, p < 0.0001 by ANOVA; n = 4 mice total, two males and two females).(J) BMDMs treated as in (H) followed by quantitative RT-PCR for relative VEGF-A165b mRNA expression (**, p < 0.009 compared with all others by ANOVA n = 4 mice total, two males and two females). Data, mean ± SD.

Fig 3: PRKAR2B regulates HIF1α expression in prostate cancer. (A‐B) Correlation analysis of the link between PRKAR2B and HIF1A expression in prostate cancer tissues; data were obtained from the TCGA cohort (A) and Ren Ji cohort (B). (C) Western blotting analysis of the effect of PRKAR2B knockdown on the HIF1A protein level in DU145 cells. (D) Western blotting analysis of the effect of PRKAR2B overexpression on the HIF1A protein level in LNCaP cells. (E) Western blotting analysis of HIF1A protein level in LNCaP and LNCaP‐AI cells. (F) Western blotting analysis of the effect of PRKAR2B knockdown on the HIF1A protein level in LNCaP‐AI cells. (G) Effects of PRKAR2B knockdown or overexpression on the HIF‐1α activity. (H) Western blotting analysis of HIF1A protein level in DU145 and PC3 cells upon H89 (50 μmol/L) treatment for 24 h. (I) Determining the effect of H89 (50 μmol/L) treatment on the HIF‐1α activity in DU145 and PC3 cells. *P < .05; **P < .01

Fig 4: Hypoxia inducible factor-1α (HIF-1α) is upregulated and functionally active in response to hypoxia. The effect of hypoxia on (A) the expression of HIF-1α protein for different time periods, and (B) activation (DNA binding) of HIF1-α. (*) denotes a significant difference compared to the normoxic group (Student’s t test, p < 0.05). (C) vascular endothelial growth factor (VEGF) production. (*) denotes groups are significantly different from each other (one way ANOVA (analysis of variance) followed by a post-hoc Tukey test, p < 0.05). (D) Expression of HIF-1α related proteins after 48 h incubation under hypoxia and normoxia. Data are represented as mean ± SD (n = 3). Representative results of three independent Western blot analyses are shown.

Fig 5: HIF1a and AhR mediate butyrate induction of IL-22 in CD4+ T cells.a WT CD4+ T cells were activated with anti-CD3/CD28 mAbs under Th1 conditions ± butyrate (0.5 mM) for 2 days (n = 3 biologically independent samples per group). RNA sequencing was performed. Hif1a, Ahr, and Prdm1 were shown in heatmap. b–f CD4+ T cells were activated with anti-CD3/CD28 mAbs ± butyrate (0.5 mM) under Th1 conditions (n = 3/group). Hif1a (b) and Ahr (c) were analyzed by qRT-PCR. HIF1a (d) and AhR (e) protein was analyzed by western blot on day 2. HIF1a activity was measured using HIF1a Transcription Factor Assay Kit (f). g Raw 264.7 cells were transduced with XRE/AhR Luciferase Reporter Gene Lentivirus, and treated ± butyrate (0.5 mM) 3 days post transduction. AhR activity was assessed by luciferase. h–j Cbir1 Tg CD4+ T cells were activated with APCs and Cbir1 peptide under Th1 conditions with butyrate (0.5 mM) ± YC-1 (5 µM) or/and CH-223191 (3 µM) for 60 h (n = 3/group). IL-22 mRNA (h) and protein (i) were measured by qRT-PCR and ELISA. j IL-22 was measured by flow cytometry on day 5. k WT and HIF1a-/- CD4+ T cells were activated with anti-CD3/CD28 mAbs ± butyrate (0.5 mM) for 5 days (n = 3/group). IL-22 was assessed by flow cytometry. l, m CD4+ T cells were activated with anti-CD3/CD28 mAbs under Th1 conditions with or without butyrate (0.5 mM), AR420626 (5 µM), or TSA (10 nM) for 60 h (n = 3/group). Hif1a (l) and Ahr (m) were measured by qRT-PCR. One representative of three independent experiments was shown (b–m). Data were expressed as mean ± SD. Statistical significance was tested by two-tailed unpaired Student t-test (b–g) or two-tailed one-way ANOVA (h–m). b **p = 0.0033 (24 h), ***p = 0.0002 (36 h), **p = 0.0032 (48 h), *p = 0.0310 (60 h); c *p = 0.0338 (24 h), **p = 0.0054 (36 h), ***p = 0.0003 (48 h), ***p = 0.0007 (60 h); d *p = 0.0178; e *p = 0.0325; f *p = 0.0273; g *p = 0.0435; h ****p < 0.0001; i ****p < 0.0001, ***p = 0.0002 (butyrate + YC-1 vs butyrate), ***p = 0.0006; j ****p < 0.0001; k **p = 0.0015, *p = 0.0325; l **p = 0.0014 (butyrate vs control) and 0.0036 (AR429626 vs control); m ****p < 0.0001, ***p = 0.0009.