Journal: Blood

Article Title: MDH1-mediated malate-aspartate NADH shuttle maintains the activity levels of fetal liver hematopoietic stem cells

doi: 10.1182/blood.2019003940

Figure Lengend Snippet: STAT3 transactivates Mdh1 expression to maintain FL-HSC activities. (A) Mdh1 luciferase reporter and different doses of Stat3 were cotransfected into 293T cells, followed by the determination of luciferase activities (n = 3). (B) ChIP assays were analyzed with 293T cells cotransfected with pGL4.27-mdh1 promoter vector and Stat3 plasmid or control plasmid. Input control and amplification of the Stat3-binding sequence of Mdh1 were determined by semiquantitative PCR. (C) Bisulfite sequencing analysis of the methylation patterns of the CpG islands in the Mdh1 promoter in CD45+ SoNar-high and -low FL hematopoietic cell fractions is shown. Each row represents a unique DNA clone; filled and open circles represent methylated and unmethylated CpGs, respectively. (D) Quantification data of methylated and unmethylated CpGs (n = 3). (E) Protein levels of pSTAT3, STAT3, and MDH1 were measured in CD45+ SoNar-high and -low FL cells. Ratios of pSTAT3/STAT3 and MDH1/actin were quantified and normalized against SoNar-high#1 cells. (F) Representative flow cytometric analysis of intracellular level of pSTAT3 of total CD45+ FL hematopoietic cells and FL-HSCs. Mean fluorescence intensity (MFI) of each group was shown (isotype control, yellow and green lines). (G) Quantification of MFI of intracellular level of pSTAT3 in panel F (n = 3). (H) Immunoblotting assay showed protein levels of pSTAT3, STAT3, and MDH1 in total CD45+ FL hematopoietic cells and FL-HSCs. Ratios of pSTAT3/STAT3 and MDH1/actin were quantified and normalized against total cells. (I) Representative flow cytometric analysis of intracellular level of pSTAT3 of CD45+ SoNar-high and -low FL-HSCs. MFI of each group is shown (isotype control, yellow and green lines). (J) Quantification of MFI of intracellular level of pSTAT3 in panel I (n = 3). (K) Protein level of MDH1 was measured in Stat3-overexpressed SoNar 32D cells and control cells by immunoblotting. Ratios of STAT3/actin and MDH1/actin were quantified and normalized against control cells. (L) Stat3-overexpressed SoNar 32D cells and control cells were evaluated for the ratios of SoNar fluorescence, and representative images are shown. Scale bar, 10 μm. (M) Quantification of the ratios of SoNar fluorescence in panel L. A total of 30 SoNar 32D cells were analyzed (n = 3). (N) Working model for the connections between FL-HSC activities and Mdh1-mediated malate-aspartate shuttle as indicated by SoNar. Data are represented as mean ± standard error of the mean. Student 2-tailed unpaired t test (G,J,M) and 2-way analysis of variance with Sidak’s multiple comparison test (D) were used for the comparison. *P < .05, ***P < .001.

Article Snippet: To test the effect of STAT3 inhibitor on MDH1 expression, purified Lin − Sca-1 + c-Kit + FL-HSCs and adult HSCs were treated with 10 or 20 μM of STAT3 inhibitor C188-9 (TargetMol) for 24 hours according to the previous study 37 and subjected to determination of pSTAT3, STAT3, or MDH1 level by western blot.

Techniques: Expressing, Luciferase, Plasmid Preparation, Control, Amplification, Binding Assay, Sequencing, Methylation Sequencing, Methylation, Fluorescence, Western Blot, Comparison

Journal: Frontiers in Immunology

Article Title: A Functionally Different Immune Phenotype in Cattle Is Associated With Higher Mastitis Incidence

doi: 10.3389/fimmu.2018.02884

Figure Lengend Snippet: Lymphocyte proteomes reveal regulation of differential expression of proteins between normal and deviant immune responders to PregSure BVD vaccination. Differential proteome analyses of PBL from control (Ctr, n = 2, shown as arithmetic means) and BNP ( n = 2, shown as arithmetic means) cows already revealed differences in protein expression patterns in constitutive expression (cE) of proteins, that shifted even stronger after (A) PWM and (B) ConA stimulation (48 h). Highly abundant proteins are presented in green and low abundant proteins in red. (C/D) In-depth analysis of part of the proteome relating to regulation of immune cells revealed some differentially (factor ≥ 2.0) expressed STAT proteins in bovine PBL after polyclonal activation with PWM (C) or ConA (D) . STAT3 and STAT5A were selectively upregulated in BNP lymphocytes (C) compared to controls due to PWM. After ConA stimulation, controls significantly upregulated STAT1 and STAT6 (D) , whereas in PBL of BNP cases STAT3 was enhanced during immune response (D) .

Article Snippet: For the inhibition assays, cells were incubated with STAT3 inhibitor (WP 1,066, Santa Cruz, Heidelberg, Germany, 50 ng/ml) 12 h before stimulation.

Techniques: Quantitative Proteomics, Control, Expressing, Activation Assay

Journal: Frontiers in Immunology

Article Title: A Functionally Different Immune Phenotype in Cattle Is Associated With Higher Mastitis Incidence

doi: 10.3389/fimmu.2018.02884

Figure Lengend Snippet: In PBL of control and BNP cows, different transcription factors were activated after ConA stimulation in vitro . (A) Representative western blot of constitutive (cE) pSTAT1 Tyr701 expression and after stimulation with ConA (ConA) in control PBL (Ctr, n = 1) and BNP PBL (BNP, n = 1). (B) Lymphocytes of vaccinated control cows (white-black striped bars, n = 5) phosphorylated STAT1 Tyr701 significantly stronger than BNP lymphocytes (black-white striped bars, n = 5) after ConA stimulation (** p < 0.01). (C) Representative western blot of constitutive (cE) pSTAT3 Tyr705 expression and after stimulation with ConA (ConA) in control PBL (Ctr, n = 1) and BNP PBL (BNP, n = 1). (D) BNP lymphocytes (black-white striped bars, n = 5) phosphorylated STAT3 Tyr705 comparatively stronger than vaccinated control lymphocytes (white-black striped bars, n = 5) in response to ConA stimulation (* p < 0.05). (A/C) Phosphorylated STAT1 Tyr701 and pSTAT3 Tyr705 signals were normalized to beta-actin and quantified using Image-J software. Protein expression is shown in mean ± SD. For statistical analyses Student‘s t test was performed.

Article Snippet: For the inhibition assays, cells were incubated with STAT3 inhibitor (WP 1,066, Santa Cruz, Heidelberg, Germany, 50 ng/ml) 12 h before stimulation.

Techniques: Control, In Vitro, Western Blot, Expressing, Software

Journal: Frontiers in Immunology

Article Title: A Functionally Different Immune Phenotype in Cattle Is Associated With Higher Mastitis Incidence

doi: 10.3389/fimmu.2018.02884

Figure Lengend Snippet: STAT3 inhibitor WP1066 selectively inhibits hyperproliferation of lymphocytes from BNP cows. STAT3 inhibitor did not inhibit proliferation of control PBL (white bar, n = 4), but significantly inhibited ConA stimulated BNP cells (black bars, n = 4, *** p < 0.001). Percentage of inhibition rate shown in mean ± SD. Student‘s t- test was used for statistical analyses.

Article Snippet: For the inhibition assays, cells were incubated with STAT3 inhibitor (WP 1,066, Santa Cruz, Heidelberg, Germany, 50 ng/ml) 12 h before stimulation.

Techniques: Control, Inhibition

Journal: Oxidative Medicine and Cellular Longevity

Article Title: IL-22 Protects against Biliary Ischemia-Reperfusion Injury after Liver Transplantation via Activating STAT3 and Reducing Apoptosis and Oxidative Stress Levels In Vitro and In Vivo

doi: 10.1155/2022/9635075

Figure Lengend Snippet: IL-22 plays a protective role by activating STAT3 in vitro. (a) Expression of STAT3 and p-STAT3 proteins in HIBEpiCs treated with IL-22 at different times. (b) Expression of STAT3 and p-STAT3 proteins after using 10 μ M and 20 μ M concentrations of STAT3 inhibitor stattic. (c) The cell cycle was detected after stattic (10 μ M) treatment, and there was no significant difference in the proportion of cells in the 4N phase compared with the CoCl 2 group. Data are shown as mean ± SD. ∗ P < 0.05. NS: no statistical difference.

Article Snippet: Subsequently, we treated the HIBEpiCs with the STAT3 inhibitor, stattic (MedChemExpress, USA), at concentrations of 10 μ M and 20 μ M for 48 h and detected the inhibition efficiency by western blot assay.

Techniques: In Vitro, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: IL-22 Protects against Biliary Ischemia-Reperfusion Injury after Liver Transplantation via Activating STAT3 and Reducing Apoptosis and Oxidative Stress Levels In Vitro and In Vivo

doi: 10.1155/2022/9635075

Figure Lengend Snippet: In vivo validation that IL-22 reduces IRI-induced apoptosis by activating STAT3. (a) The expression of STAT3 and p-STAT3 after intraperitoneal injection of RcIL-22 in rats for 1, 2, and 6 hours. (b) RcIL-22 can reduce the expression of cleaved-caspase3 and BAX protein and increase the expression of BCL2 and BCLXL protein in rats compared with the IRI group. (c) Evaluation of TUNEL expression in rat bile duct tissue sections by immunofluorescence staining. Data are shown as mean ± SD. ∗ P < 0.05.

Article Snippet: Subsequently, we treated the HIBEpiCs with the STAT3 inhibitor, stattic (MedChemExpress, USA), at concentrations of 10 μ M and 20 μ M for 48 h and detected the inhibition efficiency by western blot assay.

Techniques: In Vivo, Biomarker Discovery, Expressing, Injection, TUNEL Assay, Immunofluorescence, Staining