Journal: Journal of the American Society of Nephrology : JASN

Article Title: Progressive Renal Disease Established by Renin-Coding Adeno-Associated Virus–Driven Hypertension in Diverse Diabetic Models

doi: 10.1681/ASN.2017040385

Figure Lengend Snippet: JAK/STAT pathway is upregulated in the ReninAAV db/db uNx model of DKD. (A) Microarray analysis identified the JAK/STAT pathway as a top pathway upregulated in ReninAAV db/db uNx mice at both 8 and 12 weeks of ReninAAV treatment compared with LacZ db/db uNx controls, including Stat3, Jak2, IL6Ra, and Socs3. (B) Taqman analysis verified microarray data and showed that genes of the JAK/STAT pathway are upregulated in ReninAAV db/db uNx mice compared with LacZ controls. (C and D) Serum IL-6 and MCP-1 were upregulated in ReninAAV db/db uNx mice compared with db/m or db/db uNx controls. (E) Protein expression of pSTAT3 is upregulated in ReninAAV db/db uNx mice compared with LacZ controls. (F) Immunostaining-identified pSTAT3 is increased in renal tubule epithelium and infiltrating leukocytes compared with LacZ controls, with circles highlighting low-level detection in LacZ groups. n=5+ per group. *P<0.05 versus LacZ db/db uNx control; $P<0.05 versus db/m.

Article Snippet: Probes used were SOCS3 MM00545913_s1, IL-6 MM00446191_m1, IL6Ra MM00439653_m1, JAK1 MM00600614_m1, JAK2 MM012–8489_m1, STAT3 MM01219775_m1, and NFKB1a MM00477800_g1.

Techniques: Microarray, Expressing, Immunostaining, Control

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Progressive Renal Disease Established by Renin-Coding Adeno-Associated Virus–Driven Hypertension in Diverse Diabetic Models

doi: 10.1681/ASN.2017040385

Figure Lengend Snippet: Ingenuity Pathway Analysis of ReninAAV db/db uNx and eNOS −/− db/db models

Article Snippet: Probes used were SOCS3 MM00545913_s1, IL-6 MM00446191_m1, IL6Ra MM00439653_m1, JAK1 MM00600614_m1, JAK2 MM012–8489_m1, STAT3 MM01219775_m1, and NFKB1a MM00477800_g1.

Techniques: Activation Assay, Immunopeptidomics, Protein-Protein interactions, Inhibition

Journal:

Article Title: Cytoplasmic transport of Stat3 by receptor-mediated endocytosis

doi: 10.1093/emboj/cdf351

Figure Lengend Snippet: Fig. 2. Nuclear Stat3 DNA-binding activity is inhibited by blocking endocytosis. (A and B) DNA-binding activity of Stat3 complexes in either EGF-treated NIH-3T3/EGFR (A) or PDGF-BB-treated Balb/c-3T3 (B). Cells were transfected with empty vector control (pcDNA) or vectors encoding the endocytosis inhibitors Amph A1 or Epsin 2a, or treated with 5 µM PAO. Nuclear extracts were prepared following treatment with growth factors for the indicated times and were then subjected to EMSA analysis to detect activated Stat3 dimers bound to a 32P-labeled hSIE probe.

Article Snippet: Western blot analysis was performed using rabbit polyclonal antibodies to phosphoY 705 -Stat3 (Cell Signaling Technology, 9131) or mouse monoclonal antibodies to total Stat3 protein (BD Biosciences, 610190) to probe immunoprecipitates prepared with antibodies to total Stat3 protein (Santa Cruz Biotechnology, Stat3 C-20, sc-482) from lysates of either EGF-treated NIH-3T3/EGFR or PDGF-BB-treated Balb/c-3T3 cells.

Techniques: Binding Assay, Activity Assay, Blocking Assay, Transfection, Plasmid Preparation, Labeling

Journal:

Article Title: Cytoplasmic transport of Stat3 by receptor-mediated endocytosis

doi: 10.1093/emboj/cdf351

Figure Lengend Snippet: Fig. 3. Stat3 tyrosine phosphorylation is independent of nuclear translocation and receptor endocytosis. Western blot analyses were performed using antibodies to phosphoY705-Stat3 or total Stat3 protein to probe immunoprecipitates prepared with antibodies to total Stat3 protein from lysates of either EGF-treated NIH-3T3/EGFR (A) or PDGF-BB-treated Balb/c-3T3 (B) cells. Cells were treated with the endocytosis inhibitors followed by growth factor stimulation for the indicated times.

Article Snippet: Western blot analysis was performed using rabbit polyclonal antibodies to phosphoY 705 -Stat3 (Cell Signaling Technology, 9131) or mouse monoclonal antibodies to total Stat3 protein (BD Biosciences, 610190) to probe immunoprecipitates prepared with antibodies to total Stat3 protein (Santa Cruz Biotechnology, Stat3 C-20, sc-482) from lysates of either EGF-treated NIH-3T3/EGFR or PDGF-BB-treated Balb/c-3T3 cells.

Techniques: Translocation Assay, Western Blot

Journal:

Article Title: Cytoplasmic transport of Stat3 by receptor-mediated endocytosis

doi: 10.1093/emboj/cdf351

Figure Lengend Snippet: Fig. 4. Disruption of endocytosis with specific inhibitors blocks Stat3-dependent gene regulation. Balb/c-3T3 cells were transfected with control empty vector DNA (hatched) or expression vectors encoding the endocytosis inhibitors Amph A1 (open) or Epsin 2a (solid), the β-galactosidase vector as an internal control for transfection efficiency, and either the Stat3-specific luciferase reporter construct pLucTKS3 (A) or the Stat3-independent reporter construct pLucSRE (B). The transfected cells were harvested 48 h post-transfection following serum starvation overnight and treatment with 50 ng/ml PDGF for the times indicated. Results are shown as fold luciferase activities normalized to the β-gal internal control and are representative of three independent experiments.

Article Snippet: Western blot analysis was performed using rabbit polyclonal antibodies to phosphoY 705 -Stat3 (Cell Signaling Technology, 9131) or mouse monoclonal antibodies to total Stat3 protein (BD Biosciences, 610190) to probe immunoprecipitates prepared with antibodies to total Stat3 protein (Santa Cruz Biotechnology, Stat3 C-20, sc-482) from lysates of either EGF-treated NIH-3T3/EGFR or PDGF-BB-treated Balb/c-3T3 cells.

Techniques: Transfection, Plasmid Preparation, Expressing, Luciferase, Construct

Journal:

Article Title: Cytoplasmic transport of Stat3 by receptor-mediated endocytosis

doi: 10.1093/emboj/cdf351

Figure Lengend Snippet: Fig. 5. Stat3 localizes sequentially to endocytic vesicles at the cell membrane (A), in the cytosol (B) or at the perinuclear region (C) following PDGF treatment. (A–C) NIH-3T3 cells were treated with 50 ng/ml PDGF for 45 min at 4°C to recruit ligand-bound receptors at the cell surface, and then warmed to 37°C for the times indicated, to enable endocytosis. Staining with rabbit anti-Stat3 (red) or mouse anti-AP-2 (green) antibodies shows Stat3 in endosomes following growth factor stimulation. Images were collected using the LSM 510 program on a Zeiss confocal microscope.

Article Snippet: Western blot analysis was performed using rabbit polyclonal antibodies to phosphoY 705 -Stat3 (Cell Signaling Technology, 9131) or mouse monoclonal antibodies to total Stat3 protein (BD Biosciences, 610190) to probe immunoprecipitates prepared with antibodies to total Stat3 protein (Santa Cruz Biotechnology, Stat3 C-20, sc-482) from lysates of either EGF-treated NIH-3T3/EGFR or PDGF-BB-treated Balb/c-3T3 cells.

Techniques: Staining, Microscopy

Journal:

Article Title: Cytoplasmic transport of Stat3 by receptor-mediated endocytosis

doi: 10.1093/emboj/cdf351

Figure Lengend Snippet: Fig. 6. Stat3 co-localizes with Alexa Fluor EGF sequentially at the cell membrane (A), in the cytoplasm (B) or the perinuclear region (C) following growth factor stimulation. (A–C) NIH-3T3/EGFR cells overexpressing Stat3 were treated with 2 µg/ml Alexa Fluor EGF for 45 min at 4°C to recruit ligand-bound receptor at the cell surface, and then warmed to 37°C for the times indicated, to enable endocytosis. Immunofluorescent staining with rabbit anti-Stat3 antibodies was performed as in Figure 5. Images were collected using the LSM 510 program on a Zeiss confocal microscope.

Article Snippet: Western blot analysis was performed using rabbit polyclonal antibodies to phosphoY 705 -Stat3 (Cell Signaling Technology, 9131) or mouse monoclonal antibodies to total Stat3 protein (BD Biosciences, 610190) to probe immunoprecipitates prepared with antibodies to total Stat3 protein (Santa Cruz Biotechnology, Stat3 C-20, sc-482) from lysates of either EGF-treated NIH-3T3/EGFR or PDGF-BB-treated Balb/c-3T3 cells.

Techniques: Staining, Microscopy

Journal:

Article Title: Cytoplasmic transport of Stat3 by receptor-mediated endocytosis

doi: 10.1093/emboj/cdf351

Figure Lengend Snippet: Fig. 7. Inhibition of endocytosis blocks Stat3 translocation to the perinuclear region. Immunofluorescence analysis of NIH-3T3/EGFR cells was carried out after transfection of expression vectors encoding Stat3, Amph A1 or Epsin 2a–GFP. Antibodies to Stat3 or the HA tag of Amph A1 were used to detect localization of these proteins. Transfected cells were treated with 1 µg/ml EGF for 0 min (A), 10 min (B) and 30 min (C). Localization of Stat3 (red) and Amph A1 (green) or Epsin 2a–GFP (green) was analyzed using a Zeiss confocal microscope.

Article Snippet: Western blot analysis was performed using rabbit polyclonal antibodies to phosphoY 705 -Stat3 (Cell Signaling Technology, 9131) or mouse monoclonal antibodies to total Stat3 protein (BD Biosciences, 610190) to probe immunoprecipitates prepared with antibodies to total Stat3 protein (Santa Cruz Biotechnology, Stat3 C-20, sc-482) from lysates of either EGF-treated NIH-3T3/EGFR or PDGF-BB-treated Balb/c-3T3 cells.

Techniques: Inhibition, Translocation Assay, Immunofluorescence, Transfection, Expressing, Microscopy

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: VEGF-induced vascular permeability is reduced upon CRISPR/Cas9-mediated knockout of Stat3 in zebrafish. (A) VEGF-inducible zebrafish were crossed to Stat3 +/− (heterozygous) zebrafish to generate VEGF-inducible; Stat3 +/− double transgenic fish, which were intercrossed to generate VEGF-inducible; Stat3 −/− (KO) zebrafish. (B) CRISPR/Cas9-generated Stat3 KO zebrafish (bottom) display no overt vascular defects relative to wild-type (WT) zebrafish (top). The vascular system of 3 days post-fertilization (dpf) zebrafish was visualized by microangiography with 2000 kDa FITC-dextran. Representative images of at least three zebrafish per group are shown. Scale bars: 100 μm. (C) Microangiography using 70 kDa Texas Red-dextran permeabilizing tracer (red) and 2000 kDa FITC-dextran intersegmental vessel marker (green) was performed on 3 dpf Stat3 +/+ (negative controls without VEGF induction; left) , VEGF-induced, Stat3 +/+ (middle) and VEGF-induced, Stat3 −/− (right) zebrafish. Representative images shown were obtained using a Zeiss Apotome 2 microscope with a Fluar 5×/0.25 NA lens at room temperature (RT). Scale bars: 50 μm. (D) Quantitative analysis of vascular permeability upon VEGF stimulation in WT Stat3 +/+ ( n =30) and KO Stat3 −/− ( n =9) zebrafish. Mean±s.e.m., unpaired, two-tailed Student's t -test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Permeability, CRISPR, Knock-Out, Transgenic Assay, Generated, Marker, Microscopy, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: Endothelial cell-specific STAT3 knockout mice exhibit decreased VEGF-induced permeability. (A) Images of footpads from WT and endothelial cell-specific STAT3 knockout (STAT3 ECKO ) mice following tail vein injection with 1% Evans Blue dye and human recombinant VEGF-165 protein (2.5 µg/ml; left footpads) or PBS vehicle (right footpads) being injected into the root of the footpad. (B,C) Quantitation of Evans Blue leakage in Tie2-Cre negative; STAT3 flox/flox (WT) and Tie2-Cre positive; STAT3 flox/flox (STAT3 ECKO ) mice. n =7 mice in WT group and n =6 mice in STAT3 ECKO group. Each mouse was injected with PBS on the right anterior and posterior footpads and VEGF on the left anterior and posterior footpads. Multiple biological replicates were performed and depicted findings are representative. Mean±s.e.m., one-way ANOVA followed by Bonferroni test. A.U., arbitrary units.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Knock-Out, Permeability, Injection, Recombinant, Quantitation Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: Pharmacological inhibition of STAT3 stabilizes endothelial barrier integrity following VEGF stimulation in human endothelial cells. (A) Serum-starved human umbilical vein endothelial cells (HUVECs) were pretreated with DMSO (vehicle control) for 1 h, 30 µM AQ for 4 h, or 10 µM PYR for 1 h prior to VEGF (25 ng/ml) stimulation for 0, 2 or 5 min. Lysates were immunoblotted. Densitometry was performed, and the values below the rows of bands represent the ratio of phosphorylated protein to respective total protein. (B) Human VEGF-165 recombinant protein (VEGF; 25 ng/ml) stimulation of HUVECs promotes ZO-1 (green) disorganization at endothelial cell junctions (yellow arrows; left column; DMSO vehicle control pretreatment for 1 h prior to VEGF stimulation). ZO-1 organization is maintained upon pretreatment with 30 μM AQ for 4 h (magenta arrows; middle column) or 10 μM PYR for 1 h (magenta arrows; right column) prior to VEGF stimulation. Nuclei were stained with DAPI (blue). (C) Serum-starved human pulmonary artery endothelial cells (HPAECs) were pretreated with 10 µM PYR for 1 h prior to VEGF (25 ng/ml) stimulation for 0, 5 or 30 min. VEGF stimulation promotes disorganization of ZO-1 (green) at endothelial cell junctions (yellow arrows). ZO-1 organization is maintained when HPAECs were pretreated with PYR (magenta arrows). Nuclei were stained with DAPI (blue). (D) VEGF (25 ng/ml) stimulation of human lung microvascular endothelial cells (HMVEC-Ls) promotes ZO-1 (green) disorganization at endothelial cell junctions (yellow arrows). ZO-1 organization is maintained upon pretreatment with 20 μM PYR for 6 h prior to VEGF stimulation (magenta arrows). Nuclei were stained with DAPI (blue). At least two biological replicates were performed for each experiment depicted in A-D. Scale bars: 20 µm.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Inhibition, Recombinant, Staining

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: Suppression of STAT3 activity by pyrimethamine (PYR) inhibits VEGF-induced vascular permeability in zebrafish and mice. (A) Microangiography using 70 kDa Texas Red-dextran permeabilizing tracer (red) and 2000 kDa FITC-dextran intersegmental vessel marker (green) was performed on 3 dpf zebrafish without induced VEGF pretreated with DMSO ( n =6) or 25 μM PYR ( n =5) or 3 dpf zebrafish with induced VEGF pretreated with DMSO ( n =4) or 25 μM PYR ( n =9) for 3 days. Representative images shown were obtained using a Zeiss Apotome 2 microscope with a Fluar 5×/0.25 NA lens at RT. Scale bars: 50 μm. (B) The quantitative analysis of vascular permeability without VEGF stimulation or upon VEGF stimulation in zebrafish pretreated with DMSO or PYR. Mean±s.e.m., one-way ANOVA followed by Bonferroni test. (C) Representative images of footpads from mice treated with vehicle or PYR following tail vein injection with 1% Evans Blue and footpad injection of VEGF (2.5 μg/ml) or PBS vehicle. (D) Quantitation of Evans Blue dye leakage in C57BL/6 WT mice treated with vehicle or PYR. n =9 mice in the vehicle group and n =7 mice in the PYR group. Each mouse was injected with PBS in the right posterior footpad and VEGF in the left posterior footpad. Multiple biological replicates were performed and depicted findings are representative. Mean±s.e.m., one-way ANOVA followed by Bonferroni test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Activity Assay, Permeability, Marker, Microscopy, Injection, Quantitation Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: JAK2 phosphorylates STAT3 to transduce VEGF/VEGFR-2 signaling and promote vascular permeability. (A) To perform a STAT3 GST pull-down of VEGFR-2 and JAK2, lysates of HUVECs stimulated with serum for 30 min were used as prey. GST fusion protein STAT3 expressed in 293F cells was used as bait. GST alone served as a negative control. Binding experiments were analyzed by SDS-PAGE and visualized by immunoblotting. GST-STAT3 and GST were each detected using an anti-GST antibody. Three biological replicates were performed and depicted findings are representative. (B) JAK2 phosphorylates STAT3 in vitro . In vitro kinase assays were performed using purified human STAT3 protein and kinase active JAK2 protein. The results shown here are representative of two independent experiments. (C) Representative images of footpads from C57BL/6 WT mice treated with vehicle or JAK2 inhibitor AG490. Following tail vein injection with 1% Evans Blue dye, human VEGF-165 protein (2.5 μg/ml) or PBS vehicle was injected into the root of the footpad. After 30 min, the mice were euthanized and the footpads were excised. (D) Quantitation of Evans Blue dye leakage in C57BL/6 mice treated with vehicle or AG490. n =4 mice per group. Each mouse was injected with PBS in the right posterior footpad and VEGF in the left posterior footpad. Two biological replicates were performed and depicted findings are representative. Mean±s.e.m., one-way ANOVA followed by Bonferroni test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Transduction, Permeability, Negative Control, Binding Assay, SDS Page, Western Blot, In Vitro, Purification, Injection, Quantitation Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: STAT3 transcriptionally activates ICAM-1, a cell adhesion molecule that promotes vascular permeability. (A) Top: the pGL3-ICAM1-WT plasmid containing the human ICAM-1 promoter with a STAT3 binding site located at −115 to −107 bp. Bottom: the pGL3-ICAM1-SDM plasmid with a site-directed mutation (SDM) in the STAT3 binding site as indicated. (B) Dual luciferase assays were performed in HUVECs that were transfected with pGL3-ICAM1-WT or pGL3-ICAM1-SDM and empty vector or constitutively active STAT3. Firefly and Renilla luminescence was measured and plotted as a ratio. Mean±s.e.m., one-way ANOVA followed by Bonferroni test. n =9 technical replicates. Depicted findings are representative of three independent experiments. (C) HUVECs that had been stably transduced with lentivirus encoding STAT3-specific shRNA or control shRNA were stimulated with human VEGF-165 protein (25 ng/ml) and the lysates were immunoblotted for ICAM1, p-STAT3 (Y705) and total STAT3. Depicted data are representative of three biological replicates. (D) RNA was harvested from VEGF; Stat3 +/+ or VEGF; Stat3 −/− 3 dpf embryos for quantitative PCR. stat3 transcripts are reduced in VEGF; Stat3 −/− ( n =5) compared to VEGF; Stat3 +/+ zebrafish ( n =7). Mean±s.e.m., unpaired, two-tailed Student's t -test. (E) The expression of icam-1 was assessed by real-time quantitative PCR using RNA derived from each zebrafish embryo in the absence of VEGF induction (Stat3 +/+ , n =3; Stat3 −/− , n =2) or 8 h following VEGF induction (Stat3 +/+ , n =4; Stat3 −/− , n =3) in the heat-inducible VEGF; Stat3 mutant zebrafish. Mean±s.e.m., one-way ANOVA followed by Bonferroni test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Permeability, Plasmid Preparation, Binding Assay, Mutagenesis, Luciferase, Transfection, Stable Transfection, Transduction, shRNA, Real-time Polymerase Chain Reaction, Two Tailed Test, Expressing, Derivative Assay

Journal: PLoS ONE

Article Title: Eriocalyxin B Inhibits STAT3 Signaling by Covalently Targeting STAT3 and Blocking Phosphorylation and Activation of STAT3

doi: 10.1371/journal.pone.0128406

Figure Lengend Snippet: (A) Chemical structure of EB. (B) HepG2/STAT3-luceferase reporter cells were pretreated with EB at indicated concentrations for 2 h, and luciferase activity was measured following stimulation with IL-6 (50 ng/ml) for 5 h. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001 versus the control group without EB but with IL-6 stimulation. (C) A549 cells were pretreated with EB at indicated concentrations for 2 h before stimulation by IL-6 (10 ng/ml) for 15 min. Whole cell lysates were processed for western blot analysis with the indicated antibodies. (D) A549 cells were pretreated with 20 μM EB for various time periods (0–120 min) before stimulation by IL-6 for 15 min. Whole cell lysates were processed for western blot analysis with the indicated antibodies. (E) MDA-MB-231 and MDA-MB-468 cells were treated with EB at indicated concentrations for 2 h. Whole cell lysates were processed for western blot analysis with indicated antibodies. (F) A549 cells were cultured on coverslips in serum-free medium for 24 h. The cells were then pretreated with vehicle or 10 μM EB for 2 h, followed by 30 min stimulation with IL-6. The cells on the coverslips were then processed for immunochemical staining with an anti-STAT3 antibody or nuclei staining with Diamidino-phenyl-indole. ( G ) EB and nuclear extract from A549 cells that contained activated STAT3 proteins were preincubated for 1h prior to addition of DNA probe. The STAT3 DNA-binding activity was assessed by EMSA. The oligo band shift caused by STAT3 binding and the super shift caused by the anti-STAT3 antibody binding are indicated. (H) A549 cells were treated with EB at indicated concentrations for 2 h, followed by stimulation with IFN-γ for 15 min. Whole cell lysates were processed for western blot analysis using the antibodies as indicated.

Article Snippet: Bacteria-expressed STAT3 protein (0.2 mg/ml) (ProteinOne, Inc.) was treated with EB (20 μM) at 37°C for 2 h, and the sample was then separated by SDS-PAGE and stained with Coomassie blue.

Techniques: Luciferase, Activity Assay, Western Blot, Cell Culture, Staining, Binding Assay, Electrophoretic Mobility Shift Assay

Journal: PLoS ONE

Article Title: Eriocalyxin B Inhibits STAT3 Signaling by Covalently Targeting STAT3 and Blocking Phosphorylation and Activation of STAT3

doi: 10.1371/journal.pone.0128406

Figure Lengend Snippet: (A) A549 cells were transfected with plasmids encoding the myc-tagged wild-type, C418S, C426S, C468S, C542S, C550S, C687S, C712S, or C718S mutation of STAT3 for 24 h. The cells were then treated with EB at indicated concentrations for 2 h before stimulation with IL-6 for 15 min. Whole cell lysates were processed for western blot analysis with anti-STAT3 or anti-p-STAT3 Tyr705 antibodies. The molecular weight of the exogenous myc-tagged STAT3 is 95 kDa, while the endogenous STAT3 is 87 kDa. (B) The sequence comparison of all human STAT family members showing that the cysteine at position 712 of STAT3 is unique (highlighted in gray). (C) EB (10 mM, 3.75 μL) was incubated with the STAT3 Cys712-containing peptide FT-8 (FICVTPTT) (500 μM, 75 μL) at 37°C for 2 h, and the products were resolved by mass spectrometry. The molecular weight of peptide FT-8 is 881.3 and the molecular weight of the covalent product between EB and FT-8 is 1225.5. (D) EB (10 mM, 3.75 μL) was incubated with the peptide FT-8 (FICVTPTT) (500 μM, 75 μL) at 37°C for 2 h, and the products were analyzed by MS/MS. b3, b4, b5, b6, b7 represent the fragmented EB-containing peptides. C* represents the Cys bound by EB. (E) EB (20 mM, 3.75 μL) was incubated with peptide QP-10 (QFTKCCPPKP) (500 μM, 75 μL) at 37°C for 2 h, and the products were analyzed by mass spectrometry. (F) Computational modeling of the interaction between EB and STAT3. The crystal structure of STAT3 was obtained from PDB (Protein Data Bank). Oxygen atoms of EB were shown in red. Hydrogen bonds between Gln644, Asn647, and EB were shown in purple. The predicted distance between the α, β-unsaturated carbonyl and the thiol of Cys712 is 4.3 À as indicated.

Article Snippet: Bacteria-expressed STAT3 protein (0.2 mg/ml) (ProteinOne, Inc.) was treated with EB (20 μM) at 37°C for 2 h, and the sample was then separated by SDS-PAGE and stained with Coomassie blue.

Techniques: Transfection, Mutagenesis, Western Blot, Molecular Weight, Sequencing, Incubation, Mass Spectrometry, Tandem Mass Spectroscopy

Journal: PloS one

Article Title: Acyloxy nitroso compounds inhibit LIF signaling in endothelial cells and cardiac myocytes: evidence that STAT3 signaling is redox-sensitive.

doi: 10.1371/journal.pone.0043313

Figure Lengend Snippet: Figure 1. The acyloxy nitroso compounds, NCA and NCP, and the prototypical HNO donor, Angel’s salt, target STAT3. Chemical structures of (A) 1-Nitrosocyclohexyl acetate, NCA, (B) 1-nitrosocyclohexyl pivalate, NCP, and (C) Angeli’s salt (AS). doi:10.1371/journal.pone.0043313.g001

Article Snippet: Fluorescein-5-maleimide was from Pierce Biotechnology (Rockford, lL USA) and recombinant human STAT3 was from OriGene Technologies (Rockville, MD).

Techniques:

Journal: PloS one

Article Title: Acyloxy nitroso compounds inhibit LIF signaling in endothelial cells and cardiac myocytes: evidence that STAT3 signaling is redox-sensitive.

doi: 10.1371/journal.pone.0043313

Figure Lengend Snippet: Figure 2. NCA and NCP inhibit LIF-induced STAT3 activation in human microvascular endothelial cells. HMEC-1 were pretreated for 1 h with vehicle (0.04% v/v DMSO), (A) 100 mM NCP, or (C) 100 mM NCP. Afterwards, cells were dosed for various times with 2 ng/mL LIF. Western immunoblots of cell lysates were probed for STAT3 Y705 phosphorylation and STAT3 as a loading control. (B and D) Results were quantified and expressed as the ratio of phosphorylated STAT3 to total STAT3. **P,0.01 and ***P,0.001 vs. same time point control (n = 4); 2-way ANOVA and Bonferroni post-test. doi:10.1371/journal.pone.0043313.g002

Article Snippet: Fluorescein-5-maleimide was from Pierce Biotechnology (Rockford, lL USA) and recombinant human STAT3 was from OriGene Technologies (Rockville, MD).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Control

Journal: PloS one

Article Title: Acyloxy nitroso compounds inhibit LIF signaling in endothelial cells and cardiac myocytes: evidence that STAT3 signaling is redox-sensitive.

doi: 10.1371/journal.pone.0043313

Figure Lengend Snippet: Figure 3. NCP inhibits LIF-induced STAT3 activation in cardiac myocytes. Neonatal rat ventricular myocytes (A & B) were pretreated for 1 h with 100 mM NCP (lanes 5–8) or vehicle (0.04% v/v DMSO; lanes 1–4). Cells were then dosed with 2 ng/mL LIF for various times. Western immunoblots of cell lysates were probed for STAT3 Y705 phosphorylation and STAT3. (A) Representative immunoblot of 4 independent experiments. (B) Compiled data analysis. Adult mouse cardiac myocytes (C & D) were pretreated for 1 h with 500 mM NCP (+) or vehicle (2). Cells were then dosed with 2 ng/mL LIF for 0, 5, or 15 min. (C) Representative immunoblot of 3 independent experiments. (D) Compiled data analysis. **P,0.01 or ***P,0.001 vs. same time point control; 2-way ANOVA and Bonferroni post-test (n = 3). doi:10.1371/journal.pone.0043313.g003

Article Snippet: Fluorescein-5-maleimide was from Pierce Biotechnology (Rockford, lL USA) and recombinant human STAT3 was from OriGene Technologies (Rockville, MD).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Control

Journal: PloS one

Article Title: Acyloxy nitroso compounds inhibit LIF signaling in endothelial cells and cardiac myocytes: evidence that STAT3 signaling is redox-sensitive.

doi: 10.1371/journal.pone.0043313

Figure Lengend Snippet: Figure 4. Dose response curves for inhibition of LIF-induced STAT3 activation by NCA, NCP and Angeli’s salt (AS). HMEC-1 were pretreated (A) for 1 h with various doses (02100 mM) of NCA or NCP and the same amount of vehicle (0.04% v/v DMSO) or (B) for 30 min with 02500 mM Angeli’s salt and the same amount of vehicle (50 mM NaOH). Cells were treated for 15 min with 2 ng/mL LIF. Western immunoblots of cell lysates were probed for STAT3 Y705 phosphorylation and STAT3. Results represent 2 independent experiments for both NCA and NCP and a single experiment for Angeli’s salt. doi:10.1371/journal.pone.0043313.g004

Article Snippet: Fluorescein-5-maleimide was from Pierce Biotechnology (Rockford, lL USA) and recombinant human STAT3 was from OriGene Technologies (Rockville, MD).

Techniques: Inhibition, Activation Assay, Western Blot, Phospho-proteomics

Journal: PloS one

Article Title: Acyloxy nitroso compounds inhibit LIF signaling in endothelial cells and cardiac myocytes: evidence that STAT3 signaling is redox-sensitive.

doi: 10.1371/journal.pone.0043313

Figure Lengend Snippet: Figure 8. STAT3 possesses redox-sensitive cysteines. (A) NCA and NCP block thiolate labeling. Recombinant human STAT3 was treated with vehicle (DMSO), NCA (100 mM) or NCP (100 mM) for 1 h at room temperature and then labeled for 2 h with fluorescein-5-maleimide. Equal amounts of protein were separated by SDS-PAGE and fluorescence in the gel detected (upper panel). To ensure equal loading, Western analysis was done on each fluorescein-labeled sample. Separated proteins on nitrocellulose membranes were probed with a STAT3 antibody and imunoreactive bands quantified using the Li-COR Odyssey infrared imaging system (lower panel). Results shown are representative of 3 independent experiments. (B & C) Oxidation of STAT3 is associated with sulfenic acid formation. Purified recombinant STAT3 was immunoprecipitated and pretreated with 10 mM DTT and then treated with nothing or the oxidant o-IBZ (2.5 mM) for 1 hr at 4uC. Immunoprecipitates were processed as described under ‘‘Materials and Methods’’ to determine sulfenic acid formation (STAT3-SOH). (B) Representative blot. (C) Levels of cysteine-sulfenic acid and STAT3 were quantified by the Li-COR Odyssey Detection System. Treatment with o-IBZ resulted in a significant increase in relative sulfenic acid content. **P,0.01 vs. control, n = 3; paired Student’s t-test. doi:10.1371/journal.pone.0043313.g008

Article Snippet: Fluorescein-5-maleimide was from Pierce Biotechnology (Rockford, lL USA) and recombinant human STAT3 was from OriGene Technologies (Rockville, MD).

Techniques: Blocking Assay, Labeling, Recombinant, SDS Page, Fluorescence, Western Blot, Imaging, Purification, Immunoprecipitation, Control

Journal: PloS one

Article Title: Acyloxy nitroso compounds inhibit LIF signaling in endothelial cells and cardiac myocytes: evidence that STAT3 signaling is redox-sensitive.

doi: 10.1371/journal.pone.0043313

Figure Lengend Snippet: Figure 9. NCP enhances STAT3 glutathionylation and dimerization. HL-1 cells were treated for 30 min with vehicle (control), 500 mM NCP, 1 mM diamide, or 500 mM NCP and 1 mM diamide together. Cell extracts were prepared. (A) Equal protein amounts of cleared extracts were added to non-reducing Laemmli’s SDS-sample buffer and subjected to SDS-PAGE. Blots were probed for total STAT3 and glutathionylated protein using a rabbit and mouse antibody, respectively. Immunoreactive bands were detected using Li-COR Odyssey system and secondary antibodies that produced a red (anti-rabbit) or green (anti-mouse) signal. The overlay of the red and green signals produced an orange color. Relative levels of glutathionylated STAT3 were quantified. **P,0.01, 1-way ANOVA and Dunnett’s multiple comparison test (n = 3). (B) Cells were treated as in panel A. Cell extracts were added to non-reducing Laemmli’s SDS-sample buffer and subjected to SDS-PAGE. Blots were probed for total STAT3, which showed two bands consistent with STAT3 monomers and dimers. The intensity of the higher (dimer) band relative to the lower (monomer) band for each lane was quantified. *P,0.05 and **P,0.01, 1-way ANOVA and Newman–Keuls post-test (n = 3). doi:10.1371/journal.pone.0043313.g009

Article Snippet: Fluorescein-5-maleimide was from Pierce Biotechnology (Rockford, lL USA) and recombinant human STAT3 was from OriGene Technologies (Rockville, MD).

Techniques: Control, SDS Page, Produced, Comparison

Journal: PloS one

Article Title: Acyloxy nitroso compounds inhibit LIF signaling in endothelial cells and cardiac myocytes: evidence that STAT3 signaling is redox-sensitive.

doi: 10.1371/journal.pone.0043313

Figure Lengend Snippet: Figure 10. Oxidative stress, NCP and diamide alter the Western blot profile of STAT3 under nonreducing conditions. (A & B) Aliquots of a cleared mouse heart homogenate were incubated for 30 min with vehicle, 500 mM NCP, 1 mM diamide, or 500 mM NCP+1 mM diamide. Samples were processed for SDS-PAGE and Western blot analysis in nonreducing or reducing sample buffer. (A) Membranes were probed for STAT3 using the Li-COR Odyssey detection system. (B) Intensity of the STAT3 band in the nonreduced sample was normalized to the intensity of the band after reduction. ***P,0.001 vs. Control, 1-way ANOVA and Newman–Keuls post-test (n = 3 mouse hearts). (C) Ratio of nonreduced to reduced STAT3 in wild type (WT) and failing (Gaq) mouse hearts. STAT3 levels in mouse myocardial tissue from WT (FVB/N) and heart failure mice (Gaq overexpressing) (n = 3) were determined via immunoblot analysis under nonreducing or reducing (3.75% b-mercaptoethanol (b-ME)) conditions. Protein loads were normalized using the direct blue 71 stained membranes (DB71). *P,0.05 (Student t-test). doi:10.1371/journal.pone.0043313.g010

Article Snippet: Fluorescein-5-maleimide was from Pierce Biotechnology (Rockford, lL USA) and recombinant human STAT3 was from OriGene Technologies (Rockville, MD).

Techniques: Western Blot, Incubation, SDS Page, Control, Staining

Journal:

Article Title: Interleukin-6-Specific Activation of the C/EBP? Gene in Hepatocytes Is Mediated by Stat3 and Sp1

doi:

Figure Lengend Snippet: Binding of nuclear proteins to the Sp1(−117) site. (A) Supershift analysis using an antibody (Ab) against Sp1. Nuclear extracts were prepared from control or IL-6-treated HepG2 cells by a method that maximizes the extraction of Sp1 (see Materials and Methods). The extracts were incubated with either NRS or an Sp1-specific antibody and the δAPRE/Sp1 or δAPRE probe. The supershift generated by the Sp1 antibody in lanes 2 and 4 is indicated. (B and C) Binding of recombinant Sp1 to the δAPRE/Sp1 (B) and δAPRE (C) probes. Recombinant human Sp1 (50 ng) was used alone (lanes 5 and 10) or mixed with 8 μg of HepG2 nuclear extract (optimized for Stat protein extraction) from control or IL-6-treated cells. Stat3 antibody was added to the indicated reactions. The lower panel is a longer exposure of the top portion of the gel to emphasize the Stat3 antibody supershift complex.

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology, Inc.: Stat1 p84/p91 (E-23), Stat3 (C-20), Sp1 (PEP2), and normal rabbit immunoglobulin G (normal rabbit serum [NRS]).

Techniques: Binding Assay, Incubation, Generated, Recombinant, Protein Extraction

Journal:

Article Title: Interleukin-6-Specific Activation of the C/EBP? Gene in Hepatocytes Is Mediated by Stat3 and Sp1

doi:

Figure Lengend Snippet: Replacement of the C/EBPδ APRE with SBEs from ICAM-1 or C/EBPβ renders the promoter responsive to IFN-γ. (A) The C/EBPβ promoter contains an SBE that binds Stat1 and Stat3. A probe containing the putative SBE from the C/EBPβ promoter and Stat1- or α-Stat3-specific antibody (Ab) (lanes 3 and 4, respectively) were added to nuclear extracts from control (lane 1) or IL-6 treated (lanes 2 to 4) HepG2 cells and the reactions analyzed by EMSA. Antibody supershift species are indicated. (B) Comparison of SBE sequences from the C/EBPδ, C/EBPβ, and ICAM-1 promoters. Bases that differ from the C/EBPδ APRE sequence are underlined. (C) IL-6 and IFN-γ responsiveness of SBE swap mutants. Constructs in which the C/EBPδ APRE was exchanged with SBEs from the C/EBPβ or ICAM-1 genes were generated. These constructs and (−127)-Luc were cotransfected with pRSV β-gal into Hep3B cells and assayed for basal expression and IL-6 or IFN-γ inducibility. The values represent the average of three independent experiments. Relative basal expression was normalized to the (−127)-Luc level.

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology, Inc.: Stat1 p84/p91 (E-23), Stat3 (C-20), Sp1 (PEP2), and normal rabbit immunoglobulin G (normal rabbit serum [NRS]).

Techniques: Sequencing, Construct, Generated, Expressing

Journal:

Article Title: Interleukin-6-Specific Activation of the C/EBP? Gene in Hepatocytes Is Mediated by Stat3 and Sp1

doi:

Figure Lengend Snippet: The C/EBPδ APRE competes for binding of Stat3 to the α2-m APRE. (A) EMSA using the rat α2-m APRE, nuclear extracts (6.5 μg) from HepG2 cells, and NRS or Stat3-specific antibody (Ab) as indicated. The HepG2 cells were treated with IL-6 for 15 min. An upper complex (u) and a lower complex (l) appear in the IL-6-treated extracts (lane 3). The Stat3 antibody supershift complex (lane 4) is indicated. (B) Competition for Stat3 binding by the C/EBPδ APRE. Nuclear extracts (10 μg) from IL-6 treated HepG2 cells were incubated with Stat3-specific antibody and 10× (lanes 3 and 6), 30× (lanes 4 and 7), or 100× (lanes 5 and 8) molar excess of unlabeled wild-type (δAPRE) or mutant (δAPREm) binding site, as indicated. The rat α2-m APRE was used as a probe. The film was overexposed to emphasize the supershifted complex.

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology, Inc.: Stat1 p84/p91 (E-23), Stat3 (C-20), Sp1 (PEP2), and normal rabbit immunoglobulin G (normal rabbit serum [NRS]).

Techniques: Binding Assay, Incubation, Mutagenesis

Journal:

Article Title: Interleukin-6-Specific Activation of the C/EBP? Gene in Hepatocytes Is Mediated by Stat3 and Sp1

doi:

Figure Lengend Snippet: Selective binding of Stat3 to the C/EBPδ APRE. Nuclear extracts from control or IL-6-treated HepG2 cells were analyzed by EMSA using the wild-type or mutant C/EBPδ APRE probes and control antiserum or Stat1- or Stat3-specific antibody (Ab), as indicated. The film was overexposed to emphasize the supershift signal.

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology, Inc.: Stat1 p84/p91 (E-23), Stat3 (C-20), Sp1 (PEP2), and normal rabbit immunoglobulin G (normal rabbit serum [NRS]).

Techniques: Binding Assay, Mutagenesis

Journal:

Article Title: Interleukin-6-Specific Activation of the C/EBP? Gene in Hepatocytes Is Mediated by Stat3 and Sp1

doi:

Figure Lengend Snippet: Stat3 mediates IL-6-induced expression from the C/EBPδ promoter. (A) Stat3 but not Stat1 transactivates the C/EBPδ promoter. The (−127)-Luc construct was cotransfected into Hep3B cells with expression vectors for Stat1 or Stat3 or the parental pCDNA1 vector, together with pRSV β-gal as an internal standard, and tested for basal and IL-6-induced luciferase expression. (B) Stat3 transactivation of C/EBPδ promoter mutants. The indicated deletion and point mutants (Fig. ​(Fig.33 and ​and4)4) were cotransfected with the Stat3 expression vector into Hep3B cells and tested for basal and IL-6-inducible luciferase expression. (C) Stat3 transactivates a heterologous promoter containing the C/EBPδ APRE. The indicated TK promoter-luciferase reporter constructs (Fig. ​(Fig.8)8) were cotransfected with the Stat3 expression plasmid into Hep3B cells and tested for basal and IL-6-inducible expression. The cell extracts were assayed for luciferase and β-galactosidase activities as described in Fig. ​Fig.3.3. The values represent the averages of three to six independent experiments.

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology, Inc.: Stat1 p84/p91 (E-23), Stat3 (C-20), Sp1 (PEP2), and normal rabbit immunoglobulin G (normal rabbit serum [NRS]).

Techniques: Expressing, Construct, Plasmid Preparation, Luciferase

Journal:

Article Title: Interleukin-6-Specific Activation of the C/EBP? Gene in Hepatocytes Is Mediated by Stat3 and Sp1

doi:

Figure Lengend Snippet: Sequences and Stat binding properties of the C/EBPδ APRE and several known SBEs

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology, Inc.: Stat1 p84/p91 (E-23), Stat3 (C-20), Sp1 (PEP2), and normal rabbit immunoglobulin G (normal rabbit serum [NRS]).

Techniques: Binding Assay