Journal: The Febs Journal

Article Title: Hypoxia regulates Th17/Treg balance by altering chromatin accessibility and gene expression

doi: 10.1111/febs.70353

Figure Lengend Snippet: Hypoxia induces expression of Stat3 , Ahr , and Vdr mRNA, and increases STAT3 protein expression in induced Treg (iTreg) cells. (A) Heatmap showing selected differentially expressed genes in iTreg cells under hypoxia and normoxia conditions (H‐N). The average log fold change (logFC) is shown for genes that are statistically significant in RNA‐seq. (B) mRNA expression and chromatin accessibility of Stat3 . The assay for transposase‐accessible chromatin using sequencing (ATAC‐seq) and RNA‐seq Integrative Genomics Viewer tracks are shown from T helper 17 (Th17) and iTreg cells differentiated under normoxia (N) and hypoxia (H). (C) mRNA expression and chromatin accessibility at the last intron of Stat3 . The ATAC‐seq and RNA‐seq Integrative Genomics Viewer tracks are shown from Th17 and iTreg cells differentiated under normoxia (N) and hypoxia (H). (D) Representative western blot image of STAT3 (left) and corresponding graph showing relative protein quantification (right) in iTreg cells cultured under normoxia (N) and hypoxia (H) conditions ( n = 4). β‐actin is used as a protein loading control and sample quantification is normalized to iTreg normoxia. The protein quantification of STAT3 relative to β‐actin (STAT3 : Actin) is indicated under each lane of the representative western blot. The statistical analysis was performed using one‐sample t‐test where * P ≤ 0.05. (E) Representative dot plots (left) and quantification (right) of STAT3 and pSTAT3 from flow cytometry analysis of iTreg cells differentiated under normoxia or hypoxia conditions ( n = 2, with three technical replicates). After 3 days differentiation, iTreg cells were stimulated without (No IL‐6) and with 60 ng·mL −1 of IL‐6 (+IL‐6) for 30 min. Statistical analysis was performed using a paired t ‐test, where * P ≤ 0.05. (F) Relative Stat3 mRNA expression detected by qPCR from iTreg cells differentiated for 3 days in normoxia (N) and hypoxia (H) with and without the hypoxia‐inducible factor 1‐alpha (HIF‐1α) inhibitor (HIF‐1αi) YC‐1 (2.5 μ m n = 2, and 5 μ m n = 3). mRNA expression is normalized to the corresponding normoxia sample. Hprt was used as endogenous control. Data presented as mean ± s.e.m. (G) mRNA expression and chromatin accessibility of Vdr . The ATAC‐seq and RNA‐seq Integrative Genomics Viewer tracks are shown from Th17 and iTreg cells differentiated under normoxia (N) and hypoxia (H). (H) Relative Vdr mRNA expression detected by qPCR in iTreg cells. mRNA expression in hypoxia (H) is normalized to the corresponding normoxia (N) sample. Hprt was used as endogenous control. Data presented as mean ± s.e.m. ( n = 3). Statistical analysis was performed using a one‐sample t ‐test where * P ≤ 0.05. (I) mRNA expression and chromatin accessibility of Ahr . The ATAC‐seq and RNA‐seq Integrative Genomics Viewer tracks are shown from Th17 and iTreg cells differentiated under normoxia (N) and hypoxia (H). (J) Relative Ahr mRNA expression detected by qPCR in iTreg cells. mRNA expression in hypoxia (H) is normalized to the corresponding normoxia (N) sample. Hprt was used as endogenous control. Data presented as mean ± s.e.m. ( n = 3). Statistical analysis was performed using a one‐sample t ‐test where ‘ns’ is P > 0.05. Treg, T regulatory.

Article Snippet: For inhibitor experiments, the HIF‐1α inhibitor YC‐1 (cat. no.: ALX‐420‐025 M001; Enzo Life Sciences; Farmingdale, NY, USA) or STAT3 inhibitor Stattic (cat. no.: T6308; TargetMol; Boston, MA, USA) was added (2.5 or 5 μ m ) at the beginning of the differentiation cultures and every time media was added.

Techniques: Expressing, RNA Sequencing, Sequencing, Western Blot, Cell Culture, Control, Flow Cytometry

Journal: The Febs Journal

Article Title: Hypoxia regulates Th17/Treg balance by altering chromatin accessibility and gene expression

doi: 10.1111/febs.70353

Figure Lengend Snippet: Inhibition of HIF‐1α and STAT3 in hypoxia reduces RORγt expression but does not upregulate forkhead box P3 (FOXP3). (A) Representative dot plots of FOXP3, RORγt, and IL17 (left) and quantification of FOXP3 and RORγt (right) from flow cytometry analysis of induced Treg (iTreg) cells after 3 days differentiation under normoxia (N) or hypoxia (H) with and without the hypoxia‐inducible factor 1‐alpha (HIF‐1α) inhibitor (HIF‐1αi) YC‐1 (2.5 μ m n = 2, and 5 μ m n = 3). Data presented as mean ± s.e.m. (B) Relative mRNA expression of Vegfa ( n = 2) and Glut1 ( n = 1) detected by qPCR in iTreg cells differentiated for 3 days in normoxia or hypoxia (H) with and without the HIF‐1α inhibitor (HIF‐1αi). mRNA expression is normalized to the corresponding normoxia (N) sample. Hprt was used as endogenous control. Data presented as mean ± s.e.m. (C) Representative dot plots (left) and quantification (right) of IL‐17 and RORγt from flow cytometry analysis of T helper 17 (Th17) cells differentiated for 3 days under normoxia or hypoxia (H) conditions with and without the STAT3 inhibitor (STAT3i) Stattic (2.5 μ m and 5 μ m ). Data presented as mean ± s.e.m. ( n = 3). (D) Representative dot plots of FOXP3, RORγt, and IL17 (left) and quantification of FOXP3 and RORγt (right) from flow cytometry analysis of iTreg cells differentiated for 3 days under normoxia or hypoxia (H) conditions with and without the STAT3 inhibitor (STAT3i) Stattic (2.5 μ m n = 2 and 5 μ m n = 3). Data presented as mean ± s.e.m. Treg, T regulatory.

Article Snippet: For inhibitor experiments, the HIF‐1α inhibitor YC‐1 (cat. no.: ALX‐420‐025 M001; Enzo Life Sciences; Farmingdale, NY, USA) or STAT3 inhibitor Stattic (cat. no.: T6308; TargetMol; Boston, MA, USA) was added (2.5 or 5 μ m ) at the beginning of the differentiation cultures and every time media was added.

Techniques: Inhibition, Expressing, Flow Cytometry, Control