Journal: Cardiovascular Diabetology

Article Title: BAG3 promotes proliferation and migration of arterial smooth muscle cells by regulating STAT3 phosphorylation in diabetic vascular remodeling

doi: 10.1186/s12933-024-02216-z

Figure Lengend Snippet: Downregulation of BAG3 affects p-STAT3(705) and p-STAT3(727)

Article Snippet: Rabbit-anti-BAG3 antibody (10599-1-AP, Proteintech, WB: 1:2000; IP: 1:1000; IF: 1:100), rabbit-anti-MMP2 antibody (10373-2-AP, Proteintech, WB: 1:1000), rabbit-anti-MMP9 antibody (10375-2-AP, Proteintech, WB: 1:1000), rabbit-anti-PCNA antibody (10205-2-AP, Proteintech, WB: 1:1000), mouse-anti-Tubulin antibody (66031-1-Ig, Proteintech, WB: 1:1000), mouse-anti-GAPDH antibody (60004-1-Ig, Proteintech, WB: 1:1000), mouse-anti-Flag antibody (66008-4-Ig, Proteintech, WB: 1:1000; IP: 1:1000), rabbit-anti-JAK2 antibody (3230 S, CST, WB: 1:1000), rabbit-anti-p-JAK2 antibody (3776 S, CST, WB: 1:1000), mouse-anti-STAT3 antibody (9139 S, CST, WB: 1:1000; IP: 1:1000), rabbit-anti-p-STAT3(705) antibody (9145 S, CST, WB: 1:1000; IF: 1:100), rabbit-anti-p-STAT3(727) antibody (94,994 S, CST, WB: 1:1000; IF: 1:100), mouse-anti-ERK1/2 antibody (sc-514,302, Santa, WB: 1:1000), mouse-anti-p-ERK1/2 antibody (sc-81,492, Santa, WB: 1:1000), mouse-anti-GATA3 antibody (66400-1-Ig, Proteintech, WB: 1:1000), HRP goat-anti-rabbit IgG antibody (A21020, Abbkine, WB: 1:10000), HRP goat-anti-mouse IgG antibody (A21010, Abbkine, WB: 1:10000), Rhodamine(TRITIC) conjugated goat-anti-rabbit IgG(H + L) (SA00007-2, Proteintech, IF: 1:200), Protein A/G magnetic Beads (Cat#B23202, Biotool), AntiFade Mounting Medium (Beyotime), CCK-8 cell viability kit (B34304, Bimake), Higene transfection reagent (C1506, APPLYGEN), Jet kit (101,000,046, Polyplus), protease inhibitor (B14001, Bimake), phosphatase inhibitors (B15001, Bimake), SH-4-54 (S7337, Selleck), U0126-EtOH (S1102, Selleck).

Techniques:

Journal: Cardiovascular Diabetology

Article Title: BAG3 promotes proliferation and migration of arterial smooth muscle cells by regulating STAT3 phosphorylation in diabetic vascular remodeling

doi: 10.1186/s12933-024-02216-z

Figure Lengend Snippet: BAG3 regulates p-STAT3(705) and p-STAT3(727) through p-JAK2 and p-ERK1/2 respectively to promote VSMCs migration

Article Snippet: Rabbit-anti-BAG3 antibody (10599-1-AP, Proteintech, WB: 1:2000; IP: 1:1000; IF: 1:100), rabbit-anti-MMP2 antibody (10373-2-AP, Proteintech, WB: 1:1000), rabbit-anti-MMP9 antibody (10375-2-AP, Proteintech, WB: 1:1000), rabbit-anti-PCNA antibody (10205-2-AP, Proteintech, WB: 1:1000), mouse-anti-Tubulin antibody (66031-1-Ig, Proteintech, WB: 1:1000), mouse-anti-GAPDH antibody (60004-1-Ig, Proteintech, WB: 1:1000), mouse-anti-Flag antibody (66008-4-Ig, Proteintech, WB: 1:1000; IP: 1:1000), rabbit-anti-JAK2 antibody (3230 S, CST, WB: 1:1000), rabbit-anti-p-JAK2 antibody (3776 S, CST, WB: 1:1000), mouse-anti-STAT3 antibody (9139 S, CST, WB: 1:1000; IP: 1:1000), rabbit-anti-p-STAT3(705) antibody (9145 S, CST, WB: 1:1000; IF: 1:100), rabbit-anti-p-STAT3(727) antibody (94,994 S, CST, WB: 1:1000; IF: 1:100), mouse-anti-ERK1/2 antibody (sc-514,302, Santa, WB: 1:1000), mouse-anti-p-ERK1/2 antibody (sc-81,492, Santa, WB: 1:1000), mouse-anti-GATA3 antibody (66400-1-Ig, Proteintech, WB: 1:1000), HRP goat-anti-rabbit IgG antibody (A21020, Abbkine, WB: 1:10000), HRP goat-anti-mouse IgG antibody (A21010, Abbkine, WB: 1:10000), Rhodamine(TRITIC) conjugated goat-anti-rabbit IgG(H + L) (SA00007-2, Proteintech, IF: 1:200), Protein A/G magnetic Beads (Cat#B23202, Biotool), AntiFade Mounting Medium (Beyotime), CCK-8 cell viability kit (B34304, Bimake), Higene transfection reagent (C1506, APPLYGEN), Jet kit (101,000,046, Polyplus), protease inhibitor (B14001, Bimake), phosphatase inhibitors (B15001, Bimake), SH-4-54 (S7337, Selleck), U0126-EtOH (S1102, Selleck).

Techniques: Migration

Journal: Cardiovascular Diabetology

Article Title: BAG3 promotes proliferation and migration of arterial smooth muscle cells by regulating STAT3 phosphorylation in diabetic vascular remodeling

doi: 10.1186/s12933-024-02216-z

Figure Lengend Snippet: BAG3 modulates STAT3 phosphorylation by regulating the interaction of STAT3 with JAK2 and ERK1/2

Article Snippet: Rabbit-anti-BAG3 antibody (10599-1-AP, Proteintech, WB: 1:2000; IP: 1:1000; IF: 1:100), rabbit-anti-MMP2 antibody (10373-2-AP, Proteintech, WB: 1:1000), rabbit-anti-MMP9 antibody (10375-2-AP, Proteintech, WB: 1:1000), rabbit-anti-PCNA antibody (10205-2-AP, Proteintech, WB: 1:1000), mouse-anti-Tubulin antibody (66031-1-Ig, Proteintech, WB: 1:1000), mouse-anti-GAPDH antibody (60004-1-Ig, Proteintech, WB: 1:1000), mouse-anti-Flag antibody (66008-4-Ig, Proteintech, WB: 1:1000; IP: 1:1000), rabbit-anti-JAK2 antibody (3230 S, CST, WB: 1:1000), rabbit-anti-p-JAK2 antibody (3776 S, CST, WB: 1:1000), mouse-anti-STAT3 antibody (9139 S, CST, WB: 1:1000; IP: 1:1000), rabbit-anti-p-STAT3(705) antibody (9145 S, CST, WB: 1:1000; IF: 1:100), rabbit-anti-p-STAT3(727) antibody (94,994 S, CST, WB: 1:1000; IF: 1:100), mouse-anti-ERK1/2 antibody (sc-514,302, Santa, WB: 1:1000), mouse-anti-p-ERK1/2 antibody (sc-81,492, Santa, WB: 1:1000), mouse-anti-GATA3 antibody (66400-1-Ig, Proteintech, WB: 1:1000), HRP goat-anti-rabbit IgG antibody (A21020, Abbkine, WB: 1:10000), HRP goat-anti-mouse IgG antibody (A21010, Abbkine, WB: 1:10000), Rhodamine(TRITIC) conjugated goat-anti-rabbit IgG(H + L) (SA00007-2, Proteintech, IF: 1:200), Protein A/G magnetic Beads (Cat#B23202, Biotool), AntiFade Mounting Medium (Beyotime), CCK-8 cell viability kit (B34304, Bimake), Higene transfection reagent (C1506, APPLYGEN), Jet kit (101,000,046, Polyplus), protease inhibitor (B14001, Bimake), phosphatase inhibitors (B15001, Bimake), SH-4-54 (S7337, Selleck), U0126-EtOH (S1102, Selleck).

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Long noncoding RNA HEGBC promotes tumorigenesis and metastasis of gallbladder cancer via forming a positive feedback loop with IL-11/STAT3 signaling pathway

doi: 10.1186/s13046-018-0847-7

Figure Lengend Snippet: p-STAT3 bound to the promoter of HEGBC and activated HEGBC expression. a Schematic outline of the predicted binding site for p-STAT3 on the promoter of HEGBC . b ChIP assay in NOZ cells was performed using p-STAT3 specific antibody or negative control IgG. The bound DNA was detected using qRT-PCR with specific primers against the predicted binding sites on the promoter of HEGBC or a distal non-binding site (negative control, NC). c RIP assay in NOZ cells was performed using p-STAT3 specific antibody, STAT3 specific antibody, or negative control IgG. The bound RNA was detected using qRT-PCR with specific primers against HEGBC or TSLNC8 (positive control). d After co-transfection of renilla luciferase reporter pRL-TK with firefly luciferase reporters containing HEGBC promoter, p-STAT3 binding sites mutated HEGBC promoter, or nothing into NOZ cells, the NOZ cells were treated with 5 μM p-STAT3 inhibitor SC144 for 72 h. Luciferase activities in these NOZ cells were detected. Data are presented as the relative ratio of firefly luciferase activity to renilla luciferase activity. e After co-transfection of renilla luciferase reporter pRL-TK with firefly luciferase reporters containing HEGBC promoter, p-STAT3 binding sites mutated HEGBC promoter, or nothing into NOZ cells, the NOZ cells were treated with 20 ng/mL IL-11 for 72 h. Luciferase activities in these NOZ cells were detected. Data are presented as the relative ratio of firefly luciferase activity to renilla luciferase activity. f The expression of IL-11 in NOZ cells treated with 5 μM SC144 for 72 h was detected using qRT-PCR. g The expression of IL-11 in NOZ cells treated with 20 ng/mL IL-11 for 72 h was detected using qRT-PCR. For b - g , results are shown as mean ± s.d. of 3 independent experiments. ns, not significant, ** P < 0.01, *** P < 0.001 by Student’s t test. h The correlation between HEGBC and IL-11 expression level in 102 GBC tissues. r = 0.7996, P < 0.0001 by Pearson’s correlation analysis

Article Snippet: Where indicated, the GBC cells were treated with 5 ng/mL doxorubicin (Selleck, Houston, TX, USA) for 24 h, 5 μM p-STAT3 inhibitor SC144 (Selleck) for 72 h, or 20 ng/mL IL-11 (Gibco) for 72 h.

Techniques: Expressing, Binding Assay, Negative Control, Quantitative RT-PCR, Positive Control, Cotransfection, Luciferase, Activity Assay

Journal: Cell Death & Disease

Article Title: Hippo kinases Mst1 and Mst2 maintain NK cell homeostasis by orchestrating metabolic state and transcriptional activity

doi: 10.1038/s41419-024-06828-x

Figure Lengend Snippet: A , B Flow cytometry analysis (left) and MFI intensity (right) demonstrate the expression levels of p-STAT5, p-S6, p-AKT473 and p-Erk in NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice upon stimulation with or without IL-15 (50 ng/mL) for 30 min ( n ≥ 4). C Gene-set enrichment analysis of RNA-seq data shows the enrichment of hallmark-IL6-JAK-STAT3-signaling related genes in Mst1/2-deficient NK cells compared to WT NK cells. D Flow cytometry analysis (left) and MFI intensity (right) reveals the expression level of p-STAT3 (Ser727) in NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice upon stimulation with or without IL-15 (50 ng/mL) for 30 min ( n = 4). E ChIP-qPCR analysis demonstrated binding of p-STAT3 conserved motifs in the Tcf7 5′ regulatory region (−17.5 kb), while no binding was observed in a region without p-STAT3-binding motifs (+0.2 kb), serving as a negative control. The experiments were using sorted NK cells from WT mice with anti-p-STAT3 antibody or isotype-matched IgG. F – I WT splenocytes were treated with DMSO, Mst1 inhibitor XMU-MP-1 (1 mM, MedChemExpress) or p-STAT3 inhibitor Stattic (1 mM, MedChemExpress) for 24 h, followed by flow cytometry analysis. The representative plots (left) and summary data (right) show the expression level of p-STAT3 (F), TCF1 ( G ), total cellular ROS ( H ) and the percentage of 7AAD + NK cells ( I ) ( n = 4). J Schematic illustration of Mst1/2 regulate NK cell survival and function via controlling metabolic state and transcriptional activity. Data A , B and D – I are representative of three independent experiments with similar results.

Article Snippet: F – I WT splenocytes were treated with DMSO, Mst1 inhibitor XMU-MP-1 (1 mM, MedChemExpress) or p-STAT3 inhibitor Stattic (1 mM, MedChemExpress) for 24 h, followed by flow cytometry analysis.

Techniques: Flow Cytometry, Expressing, RNA Sequencing, ChIP-qPCR, Binding Assay, Negative Control, Activity Assay

Journal: Journal of molecular and cellular cardiology

Article Title: Angiotensin II-induced TLR4 mediated abdominal aortic aneurysm in apolipoprotein E knockout mice is dependent on STAT3.

doi: 10.1016/j.yjmcc.2015.08.014

Figure Lengend Snippet: Fig. 1. The STAT3 inhibitor, S3I-201, decreases the incidence and severity of AAA formation. A. Effect of AngII on pSTAT3 and tSTAT3 in aneurysmal tissues determined by western blotting. B. Densitometric analysis of data in A normalized to GAPDH (*p b 0.05, **p b 0.01 vs. control, n = 4 per group). C. S3I-201 significantly reduced the incidence of AAA formation from 84% (AngII) to 40% (p b 0.05, n = 19 and 15), D. S3I-201 significantly reduced the severity of AAA based on the classification of aneurysms. E. The external diameter of the aorta was also significantly decreased by S3I-201 treatment (##p b 0.01 vs. placebo, n = 19 and 15 respectively), F & G. S3I-201 decreased AngII-induced pSTAT3/tSTAT3 determined by western blot (**p b 0.01 vs. control, ##p b 0.01 vs. placebo, n = 4).

Article Snippet: Placebo, STAT3 inhibitor S3I-201 (5 mg/kg, i.p., every other day, Selleckchem) or TLR4 antagonist Eritoran (0.3 mg/kg, i.v., every other day, Eisai Inc.) were given for 28 days.

Techniques: Western Blot, Control

Journal: Journal of molecular and cellular cardiology

Article Title: Angiotensin II-induced TLR4 mediated abdominal aortic aneurysm in apolipoprotein E knockout mice is dependent on STAT3.

doi: 10.1016/j.yjmcc.2015.08.014

Figure Lengend Snippet: Fig. 6. The TLR4 inhibitor Eritoran decreases the incidence of AngII-induced AAAs, the activation of STAT3 and M1/M2 ratio. A & B. Eritoran significantly reduced the incidence of AngII- induced AAA formation and the external diameter of the aortas (*p b 0.05 vs. placebo, n = 9 and 10 per group). C & D. Eritoran decreased the gelatinolytic activities of MMP2 and MMP9 in AngII-treated ApoE−/−mice measured by zymography in aortic homogenates (**p b 0.01 vs. control, ##p b 0.01 vs. placebo, n = 4). E & F. The activation of STAT3 induced by AngII was significantly decreased by Eritoran, as determined by western blot analysis of aortic homogenates (*p b 0.05 vs. control, #p b 0.05 vs. placebo, n = 4). G. The effect of Eritoran on iNOS, Ym-1 and the ratio of iNOS/Ym-1 mRNA levels, as measured by qPCR (n = 4, *p b 0.05, vs. control, **p b 0.01 vs. control, ##p b 0.01 vs. placebo).

Article Snippet: Placebo, STAT3 inhibitor S3I-201 (5 mg/kg, i.p., every other day, Selleckchem) or TLR4 antagonist Eritoran (0.3 mg/kg, i.v., every other day, Eisai Inc.) were given for 28 days.

Techniques: Activation Assay, Zymography, Control, Western Blot

Journal: Journal of molecular and cellular cardiology

Article Title: Angiotensin II-induced TLR4 mediated abdominal aortic aneurysm in apolipoprotein E knockout mice is dependent on STAT3.

doi: 10.1016/j.yjmcc.2015.08.014

Figure Lengend Snippet: Fig. 7. Abdominal aortas from patients with AAA demonstrate increased levels of TLR4, pSTAT3 and tSTAT3 compared to normal donor aortas. A & B. Western blot analysis of TLR4, pSTAT3, and tSTAT3 in aortic tissue taken at the time of aneurysmal repair and aortas from normal organ donors (*p b 0.05 vs. control, n = 4). C. Schematic representation of proposed pathway for the link between STAT3 and TLR4 in AngII-induced of AAA formation.

Article Snippet: Placebo, STAT3 inhibitor S3I-201 (5 mg/kg, i.p., every other day, Selleckchem) or TLR4 antagonist Eritoran (0.3 mg/kg, i.v., every other day, Eisai Inc.) were given for 28 days.

Techniques: Western Blot, Control

Journal: Oncology reports

Article Title: Taurolidine promotes cell apoptosis by enhancing GRIM‑19 expression in liver cancer.

doi: 10.3892/or.2018.6711

Figure Lengend Snippet: Figure 4. GRIM‑19 regulates HepG2 cell proliferation and apoptosis via a STAT3‑dependent pathway. The expression levels of proliferation‑ and apop- tosis‑associated molecules, STAT3, p‑STAT3, cyclin D1, Bcl‑2 and Bax, were detected by western blotting. (A) Expression of p‑STAT3, cyclin D1 and Bcl‑2 was increased, and that of Bax was decreased, in the GRIM‑19 siRNA group, whereas TRD treatment had the opposite effect, inhibiting the expression of p‑STAT3, cyclin D1 and Bcl‑2, and increasing that of Bax, compared with the control group. (B) High expression levels of GRIM‑19 and Bax were detected in the TRD and TRD + niclosamide groups, which were accompanied by low levels of cyclin D1, Bcl‑2 and‑STAT3. Conversely, low expression levels of GRIM‑19 and Bax, and high expression levels of cyclin D1, Bcl‑2 and p‑STAT3 were detected in the TRD + GRIM‑19 siRNA group. (C) Schematic diagram. Data are representative images or expressed as the means ± standard deviation of each group from three separate experiments. Bax, Bcl‑2‑associated X protein; Bcl‑2, B‑cell lymphoma 2; GRIM‑19, retinoic‑interferon‑induced mortality 19; p‑STAT3, phosphorylated‑STAT3; siRNA, small interfering RNA; STAT3, signal transducer and activator of transcription 3; TRD, taurolidine.

Article Snippet: GRIM-19 knockdown cells were pretreated with niclosamide (p-STAT3 inhibitor, 10 μM; 50-65-7; Selleck Chemicals, Houston, TX, USA) for 30 min, after which, 100 μM TRD (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was added for 24 h. MTT assay.

Techniques: Expressing, Western Blot, Control, Standard Deviation, Small Interfering RNA

Journal: eBioMedicine

Article Title: Blockade of STAT3/IL-4 overcomes EGFR T790M- cis -L792F-induced resistance to osimertinib via suppressing M2 macrophages polarization

doi: 10.1016/j.ebiom.2022.104200

Figure Lengend Snippet: EGFR 19Del/T790M- cis -L792F stimulated JAK/STAT3/IL-4 signaling. (a). Heatmap of cytokine antibody array in H1975 control and H1975 L792F cis cells. (b-c). IL-4 expression was detected by western blot (b) and ELISA analyses (c) in the H1975 control, H1975 L792F cis , PC-9 control, and PC-9 L792F cis cells ( n =3, p= 0.016 or 0.007, respectively. Statistical differences were evaluated using t test). (d). Signal pathway enrichment in the H1975 L792F cis cells compared to the H1975 control cells. (e). Western blot shows ERK, p-ERK Thr202/Tyr204, AKT, p-AKT Ser473, JAK, p-JAK Tyr1034/1035, STAT3, p-STAT3 Tyr705, p-STAT3 Ser727, STAT6, and p-STAT6 Try641 expression in the H1975 control, H1975 L792F cis , PC-9 control, and PC-9 L792F cis cells. (f). Schematic depiction of the IL4 promoters (−1000 to 1 base pair, black line) with STAT3 transcription factor binding sites (TFBs). (g-h). ChIP assay using p-STAT3 antibody and IgG as internal controls. PCR (g) and RT-qPCR (h, n=3, p= 0.006, statistical differences were evaluated using t test.) were performed to amplify p-STAT3 TFBs of IL4 promoter in the ChIP product. (i). IL4 promoter activity of luciferase reporter in the PC-9 control and PC-9 L792F cis cells with wild-type or mutant p-STAT3-binding sequences ( n =3, p= 0.00001 or 0.00002, statistical differences were evaluated using t test). PRL-TK plasmid was used as an internal control. Data are presented as means ± standard deviation.

Article Snippet: Mice were treated with osimertinib (10 mg/kg, p.o., 5 days a week) or a vehicle (osimertinib diluted solution) or STAT3 inhibitor SH-4-54 (10 mg/kg p.o. twice a week, Selleck) or IL-4 inhibitor dupilumab (10 mg/kg p.o. twice a week, MedChem Express).

Techniques: Ab Array, Control, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay, Quantitative RT-PCR, Activity Assay, Luciferase, Mutagenesis, Plasmid Preparation, Standard Deviation

Journal: eBioMedicine

Article Title: Blockade of STAT3/IL-4 overcomes EGFR T790M- cis -L792F-induced resistance to osimertinib via suppressing M2 macrophages polarization

doi: 10.1016/j.ebiom.2022.104200

Figure Lengend Snippet: Inhibition of STAT3/IL-4 signaling restored sensitivity to osimertinib. (a). Western blot shows STAT3 expression in the PC-9 L792F cis cells transfected with scramble, sg STAT3 #1, sg STAT3 #2, or sg STAT3 #3, and IL-4 expression in the PC-9 L792F cis cells transfected with scramble, sg IL4 #1, sg IL4 #2, or sg IL4 #3. (b-c) Representative cell viability curve and osimertinib IC50 value in the PC-9 L792F cis cells transfected with scramble, sg STAT3 #2 or sg STAT3 #3 (b, n =3, p= 0.001 or 0.0007, respectively. Statistical differences were evaluated using t test.), and in the PC-9 L792F cis cells transfected with scramble, sg IL4 #2 or sg IL4 #3 (c, n=3, p= 0.0006 or 0.001, respectively. Statistical differences were evaluated using t test.), under osimertinib treatment for 72 h. (d-e). Tumor growth curves and tumor images of the PC-9 L792F cis -scramble, PC-9 L792F cis -sg STAT3 #2, PC-9 L792F cis -sg STAT3 #3 (d) ( n= 5, p= 0.01 or 0.001, respectively. Statistical differences were evaluated using t test), and PC-9 L792F cis - scramble, PC-9 L792F cis - IL4 #2, and PC-9 L792F cis - IL4 #3 (e) ( n= 6, p= 1E-08 or 9E-08, respectively. Statistical differences were evaluated using t test) in the nude mice with osimertinib (10 mg/kg, 5 days a week) treatment. Scale bar, 2 cm. (f-h). Growth curve (f), tumor images (g), and tumor weight (h, n= 5, p= 0.0012 or 0.0028, respectively. Statistical differences were evaluated using t test.) of PC-9 L792F cis xenografts treated with osimertinib (10 mg/kg, 5 days a week), osimertinib (10 mg/kg, 5 days a week) + STAT3 inhibitor (SH-4-54, [10 mg/kg, twice a week]), and osimertinib (10 mg/kg, 5 days a week) + IL-4 receptor antibody (dupilumab, 10 mg/kg, twice a week). Scale bar, 2 cm. (i). Inhibitory rates of SH-4-54 and dupilumab for PC-9 L792F cis xenografts. (j-k). Images of H&E (upper panel in j), immunohistochemistry staining for CD206 (down panel in j) and CD206 + /F4-80 + macrophages fractions in PC-9 L792F cis xenografts treated with osimertinib, osimertinib + SH-4-54, and osimertinib + dupilumab (k). Scale bar, 50 μm.

Article Snippet: Mice were treated with osimertinib (10 mg/kg, p.o., 5 days a week) or a vehicle (osimertinib diluted solution) or STAT3 inhibitor SH-4-54 (10 mg/kg p.o. twice a week, Selleck) or IL-4 inhibitor dupilumab (10 mg/kg p.o. twice a week, MedChem Express).

Techniques: Inhibition, Western Blot, Expressing, Transfection, Immunohistochemistry, Staining

Journal: Hepatology (Baltimore, Md.)

Article Title: Endothelial YAP/TEAD1-CXCL17 signaling recruits myeloid-derived suppressor cells against liver ischemia-reperfusion injury

doi: 10.1097/HEP.0000000000000773

Figure Lengend Snippet: Intrahepatic MDSCs dampened IRI-induced damage through suppressing M1 macrophage polarization. Mice were treated with αGr-1 antibody, exogenous MDSCs transfer, or isotype IgG control at the onset of reperfusion. After 12 hours, mice were killed and samples were collected for analysis. (A) Representative images of H&E staining for liver sections from sham or liver IRI–mediating mice with depletion or adoptive transfer of MDSCs (×100; ×200). The scale bar is 100 or 50 μm, respectively. (B) Quantification of serum ALT and AST in mice under different treatments. (C) Intrahepatic levels of proinflammatory cytokines IL-1β, IL-6, and TNF-α in mice under different treatments. (D) Flow cytometry analysis and quantification of the proportions as well as CD86 (M1 polarization marker) expression of macrophages under conditions of depletion or adoptive transfer of MDSCs. (E, F) Primary peritoneal macrophages were polarized by HMGB1 (1 μg/mL) and cocultured with sorted MDSCs (cell ratio 1:3) in vitro for 24 hours, along with the treatment of anti-IL-10 or anti-TGF-β neutralizing antibody (10 μg/mL), ARG1 inhibitor l -Norvaline (20 mM) or STAT3 inhibitor Stattic (5 μM). Expression levels of CD86 and proinflammatory cytokine genes ( Il6 , Tnf , and Il1b ) in macrophages were tested. Data are shown as mean±SD. n=4 per group. ANOVA test, * p <0.05, ** p <0.01, *** p <0.001, ns, no significance. Abbreviations: IgG, immunoglobulin G; IRI, ischemia-reperfusion injury; MDSC, myeloid-derived suppressor cell.

Article Snippet: Stattic (STAT3 inhibitor, 5 mg/kg, i.p., MCE) or DMSO as control was administrated 12 hours before the onset of ischemia.

Techniques: Control, Staining, Adoptive Transfer Assay, Flow Cytometry, Marker, Expressing, In Vitro, Derivative Assay

Journal: Genes & Diseases

Article Title: Autophagy-induced NR2F1 activation promotes the apoptosis of lens epithelial cells and facilitates cataract-associated fibrosis through targeting STAT3

doi: 10.1016/j.gendis.2025.101549

Figure Lengend Snippet: NR2F1 directly binds to STAT3 and regulates the expression of p-STAT3. (A, B) The protein levels and quantification of JAK1, p-STAT3, SMAD2, and MYD88 in TGF-β1-treated SRA01/04 cells with or without Sh-NR2F1. n = 3 per group; mean ± standard deviation; ns, >0.05; ∗∗ p < 0.01; unpaired student's t -test. (C, D) The protein level and quantitative chart of p-STAT3 in AAV-NC or AAV-NR2F1 ASC mice. n = 3 per group; mean ± standard deviation; ∗ p < 0.01; unpaired student's t -test. (E) The motif of NR2F1 predicted by the JASPAR website. (F) A dual-luciferase vector. (G) Mutant and wild-type STAT3 plasmids consequences. (H) The dual-luciferase assay for NR2F1 and STAT3 promoter. n = 3 per group; mean ± standard deviation; ns, >0.05; ∗ p < 0.05; unpaired student's t -test. (I, J) The protein level of p-STAT3 in TGF-β1-induced SRA01/04 cells following NR2F1 agonist treatment at 0.5 or 1 μM. n = 3 per group; mean ± standard deviation; ns > 0.05; ∗ p < 0.05; one-way ANOVA. (K) Structure of p-STAT3 specific inhibitor NSC 74859. (L, M) The protein expression and quantification of p-STAT3, STAT3, JAK1, FN1, VIM, and α-SMA in TGF-β1-induced SRA01/04 cells treated with the specific P-STAT3 inhibitor. n = 3 per group; mean ± standard deviation; ns, >0.05, ∗ p < 0.05, ∗∗ p < 0.01; one-way ANOVA. (N, O) The protein expression of the apoptosis-related markers BAX and CASP3 in TGF-β1-induced SRA01/04 cells treated with NSC 74859. n = 3 per group; mean ± standard deviation; ∗ p < 0.05; one-way ANOVA. NR2F1, nuclear receptor subfamily 2 group F member 1; STAT3, signal transducer and activator of transcription 3; p-STAT3, phosphorylated STAT3; ASC, anterior subcapsular cataract; FN1, fibronectin 1; α-SMA, α-smooth muscle actin; VIM, vimentin; BAX, Bcl-2-associated X; CASP3, caspase 3; TGF-β1, transforming growth factor-β1; JAK1, Janus kinase 1; SMAD2, SMAD family member 2; AAV, adeno-associated adenovirus.

Article Snippet: Additionally, the P-STAT3 inhibitor NSC 74859 (MCE, Shanghai, China) was added 24 h before TGF-β1 treatment.

Techniques: Expressing, Standard Deviation, Luciferase, Plasmid Preparation, Mutagenesis