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

Journal: Journal of Inflammation Research

Article Title: Anti-Inflammatory Regulatory Role of Signal Transducer and Activator of Transcription 3 Phosphorylation in Regulating Hypersensitivity Responses to Echinococcus granulosus Hydatid Cyst Fluid

doi: 10.2147/JIR.S509286

Figure Lengend Snippet: The inhibitory effect of different concentrations of inhibitors on STAT3 phosphorylation.

Article Snippet: The STAT3 inhibitors Stattic and JSI-124 were procured from Shanghai Haoyuan Chemexpress Co., Ltd. Antibodies for STAT3, phosphorylated p-STAT3 (S727), p-STAT3 (Y705), and ACTIN, as well as secondary antibodies, were purchased from Wuhan ABclonal Biotechnology Co., Ltd. High-resolution rapid electrophoresis liquid and ice-free rapid transfer liquid were obtained from Wuhan Servicebio Technology Co., Ltd. Immunoglobulin E (IgE) was purchased from Sigma-Aldrich, USA.

Techniques: Phospho-proteomics

Journal: International Journal of Molecular Sciences

Article Title: Resveratrol Is a Natural Inhibitor of Human Intestinal Mast Cell Activation and Phosphorylation of Mitochondrial ERK1/2 and STAT3

doi: 10.3390/ijms22147640

Figure Lengend Snippet: Phosphorylation of signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase 1/2 (ERK1/2) in hiMC following treatment with stattic, nobiletin, and resveratrol. Western blot analyses of ( A ) phosphorylated STAT3 ( n = 5) and ( B ) phosphorylated ERK1/2 ( n = 4) in hiMC. HiMCs were treated with 60 μM of stattic (S), 45 μM of nobiletin (N), 50 μM of resveratrol (R), or corresponding concentrations of the vehicle DMSO (control) before stimulation by FcεRI crosslinking using 100 ng/mL of mAb 22E7 for 5 min. Representative pictures and densitometric analyses in relation to stimulated control are shown. Values are mean ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: To analyze STAT3 activation, hiMCs were pre-incubated for 20 min with 60 μM STAT3 inhibitor stattic (Merck) prior to stimulation by FcεRI crosslinking.

Techniques: Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Resveratrol Is a Natural Inhibitor of Human Intestinal Mast Cell Activation and Phosphorylation of Mitochondrial ERK1/2 and STAT3

doi: 10.3390/ijms22147640

Figure Lengend Snippet: Phosphorylation of STAT3 and ERK1/2 in nuclear and mitochondrial fractions of hiMC following treatment with resveratrol. Western blot analyses of two independent experiments of ( A ) phosphorylated ERK1/2 and ( B ) phosphorylated STAT3 after subcellular fractionation in pure mitochondria (Mito), cytosol fraction (Cyto), and crude nuclear fraction (Nuc), respectively. HiMCs were treated with 50 μM of resveratrol (R), or corresponding concentrations of the vehicle DMSO (control), and stimulated by FcεRI crosslinking with 100 ng/mL of mAb 22E7 for 5 min. Exemplary pictures and densitometric analyses in relation to stimulated control for Western blots of phosphorylated ERK1/2 and total ERK1/2, and phosphorylated STAT3 and total STAT3, are shown. Values are mean ± SEM ( n = 2).

Article Snippet: To analyze STAT3 activation, hiMCs were pre-incubated for 20 min with 60 μM STAT3 inhibitor stattic (Merck) prior to stimulation by FcεRI crosslinking.

Techniques: Western Blot, Fractionation

Journal: Frontiers in Pharmacology

Article Title: Tirbanibulin Attenuates Pulmonary Fibrosis by Modulating Src/STAT3 Signaling

doi: 10.3389/fphar.2021.693906

Figure Lengend Snippet: The impact of KX-01 on pulmonary protein expression. (A, B) : The expression of HIF-1α, p-Src; (C, D) : p-STAT3, STAT3 and p-STAT3/STAT3 was analyzed. BLM: bleomycin. Data are means ± SD ( n = 3). # p < 0.01 vs. sham; * p < 0.01 vs. BLM. Data were compared via ANOVAs with Dunnett’s test.

Article Snippet: The STAT3 inhibitor Stattic (purity>98%, C 8 H 5 NO 4 S, CAS No: 19983-44-9) was from the Jiangsu Aikang Biomedicine Company (China).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Tirbanibulin Attenuates Pulmonary Fibrosis by Modulating Src/STAT3 Signaling

doi: 10.3389/fphar.2021.693906

Figure Lengend Snippet: The impact of KX-01 on pulmonary α-SMA and p-STAT3 immunohistochemical staining. (A) and (4C) : Immunohistochemical staining was used to assess pulmonary α-SMA; (B–D) : Immunohistochemical staining was used to assess pulmonary p-STAT3 expression following BLM treatment with or without KX-01 administration. BLM: bleomycin. Data are means ± SD. # p < 0.01 vs. sham; * p < 0.01 vs. BLM. Data were compared via ANOVAs with Dunnett’s test.

Article Snippet: The STAT3 inhibitor Stattic (purity>98%, C 8 H 5 NO 4 S, CAS No: 19983-44-9) was from the Jiangsu Aikang Biomedicine Company (China).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Frontiers in Pharmacology

Article Title: Tirbanibulin Attenuates Pulmonary Fibrosis by Modulating Src/STAT3 Signaling

doi: 10.3389/fphar.2021.693906

Figure Lengend Snippet: The impact of KX-01 on L929 cell proliferation and protein expression. (A) An EdU incorporation assay was used to assess L929 cell proliferation following CoCl 2 treatment with or without KX-01 for 72 h. (B–C) : The expression of α-SMA, collagen I, and collagen III were assessed; (D, E) : The expression of HIF-1α, p-Src (D–E); (F, G) : The expression of p-STAT3 and STAT3, and the ratio of p-STAT3/STAT3 was assessed. Data are means ± SD ( n = 3). # p < 0.01 vs. control; * p < 0.01 vs. CoCl 2 . Data were compared via ANOVAs with Dunnett's tst.

Article Snippet: The STAT3 inhibitor Stattic (purity>98%, C 8 H 5 NO 4 S, CAS No: 19983-44-9) was from the Jiangsu Aikang Biomedicine Company (China).

Techniques: Expressing, Control

Journal: Frontiers in Pharmacology

Article Title: Tirbanibulin Attenuates Pulmonary Fibrosis by Modulating Src/STAT3 Signaling

doi: 10.3389/fphar.2021.693906

Figure Lengend Snippet: The impact of KX-01 on L929 cell proliferation and protein expression in the presence of Roxadustat. (A) An EdU incorporation assay was used to assess L929 cell proliferation following CoCl 2 treatment with or without KX-01 or ROT for 72 h. (B, C) : The expression of α-SMA, collagen I, and collagen III were assessed; (D, E) : The expression of HIF-1α, p-Src, p-STAT3 and STAT3, and the ratio of p-STAT3/STAT3 were assessed. ROT: Roxadustat. Data are means ± SD ( n = 3). # p < 0.05, ## p < 0.01 vs. control; * p < 0.05, ** p < 0.01 vs. CoCl 2 . Data were compared via ANOVAs with Dunnett's test.

Article Snippet: The STAT3 inhibitor Stattic (purity>98%, C 8 H 5 NO 4 S, CAS No: 19983-44-9) was from the Jiangsu Aikang Biomedicine Company (China).

Techniques: Expressing, Control

Journal: Frontiers in Pharmacology

Article Title: Tirbanibulin Attenuates Pulmonary Fibrosis by Modulating Src/STAT3 Signaling

doi: 10.3389/fphar.2021.693906

Figure Lengend Snippet: The impact of KX-01 on L929 cell proliferation and protein expression in the presence of Stattic. (A) An EdU incorporation assay was used to assess L929 cell proliferation following CoCl 2 treatment with or without KX-01 or Stattic for 72 h. (B, C) : The expression of α-SMA, collagen I, and collagen III were assessed; (D, E) : The expression of HIF-1α, p-Src, p-STAT3 and STAT3, and the ratio of p-STAT3/STAT3 were assessed. Data are means ± SD ( n = 3). # p < 0.01 vs. control; * p < 0.05, ** p < 0.01 vs. CoCl 2 . Data were compared via ANOVAs with Dunnett's test.

Article Snippet: The STAT3 inhibitor Stattic (purity>98%, C 8 H 5 NO 4 S, CAS No: 19983-44-9) was from the Jiangsu Aikang Biomedicine Company (China).

Techniques: Expressing, Control