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: Brain, behavior, and immunity

Article Title: Increased IL-6 expression in astrocytes is associated with emotionality, alterations in central amygdala GABAergic transmission, and excitability during alcohol withdrawal

doi: 10.1016/j.bbi.2019.08.185

Figure Lengend Snippet: Summary of protein activation/expression in hippocampus

Article Snippet: Antibodies to the following proteins were used in these studies: a monoclonal rabbit antibody produced in rabbits by immunizing with a fusion protein corresponding to a sequence in the carboxy-terminal of mouse STAT3 protein (AB#4904; 1:1000; Cell Signaling Technologies); a purified rabbit polyclonal antibody produced in rabbits by immunizing with a synthetic phospho-peptide corresponding to the residues surrounding Tyr705 of mouse STAT3 (AB#9131; 1:1000; Cell Signaling Technologies); A purified rabbit polyclonal antibody produced by immunizing rabbits with a synthetic peptide derived from human GAD65/GAD67 (#PA5–38102, 1–2000, Invitrogen); a purified mouse monoclonal antibody to gamma-aminobutyric acid receptor (GABA A R) subunit alpha-5 (GABA A R alpha-5) produced by immunizing mice with a fusion protein containing a sequence from the cytoplasmic domain (amino acids 368–419) of human GABA A R alpha-5 (Clone N415/24, 1–500, UC Davis/NIH NeuroMab Facility); a purified mouse monoclonal antibody to gamma-aminobutyric acid receptor subunit alpha-1 (GABA A R alpha-1) produced by immunizing mice with a fusion protein (amino acids 355–394) from mouse GABA A R alpha-1 (clone N95/35, 1–500, UC Davis/NIH NeuroMab Facility); a purified mouse monoclonal antibody to a synthetic peptide (amino acids 411–422) of human glial fibrillary acidic protein (GFAP) (#75–240, 1–10,000, UC Davis/NIH NeuroMab Facility); a purified rabbit polyclonal antibody produced by immunizing rabbits with a synthetic peptide derived from the sequence of human enolase-2 (neuron specific enolase) (AB#9536, 1–1000, Cell Signaling Technology); a monoclonal antibody to beta-actin produced by immunizing mice with a synthetic peptide corresponding to a sequences in the amino-terminal of human beta-actin (AB#3700, 1:10,000; Cell Signaling Technology); a monoclonal antibody to beta-actin produced by immunizing rabbits with a synthetic peptide corresponding to a sequences in the amino-terminal of human beta-actin (AB#4970, 1:5,000; Cell Signaling Technology).

Techniques: Activation Assay

Journal: Brain, behavior, and immunity

Article Title: Increased IL-6 expression in astrocytes is associated with emotionality, alterations in central amygdala GABAergic transmission, and excitability during alcohol withdrawal

doi: 10.1016/j.bbi.2019.08.185

Figure Lengend Snippet: Protein activation/expression is altered in hippocampus from IL-6 tg +/− and IL6 tg +/+ mice. A. Relative levels of pSTAT3 and STAT3 in hippocampus from IL-6 tg −/−, IL-6 tg +/− and IL-6 tg +/+ mice. B,C. STAT3 (D) and pSTAT3 (C) levels in hippocampus from alcohol naïve and alcohol exposed/withdrawn IL-6 tg −/−, IL-6 tg +/− and IL-6 tg +/+ mice. D,E. Relative levels of GFAP in hippocampus from alcohol naïve and alcohol exposed/withdrawn IL-6 tg −/−, IL-6 tg +/− and IL-6 tg +/+ mice. For these and other graphs of Western blot data, parametric data are represented in bar graphs and nonparametric data are represented in box graphs. Representative samples of Western blots are shown below the respective bar or box graph. The top band of the Western blot is the protein of interest and the bottom band is beta-actin. N= naïve, A = exposed/withdrawn. Solid bars represent alcohol naïve samples and stripped bars represent alcohol exposed/withdrawn samples. Data analyzed by ANOVA are expressed as the mean ± SEM (bar graphs). For non-parametric data, median, maximum, and minimum values are shown (box plots; open circles are data points in box plots). * = significantly different from IL-6 tg −/− of the same treatment group., # = significantly from alcohol naïve of the same genotype. @= significantly different from IL-6 +/−.

Article Snippet: Antibodies to the following proteins were used in these studies: a monoclonal rabbit antibody produced in rabbits by immunizing with a fusion protein corresponding to a sequence in the carboxy-terminal of mouse STAT3 protein (AB#4904; 1:1000; Cell Signaling Technologies); a purified rabbit polyclonal antibody produced in rabbits by immunizing with a synthetic phospho-peptide corresponding to the residues surrounding Tyr705 of mouse STAT3 (AB#9131; 1:1000; Cell Signaling Technologies); A purified rabbit polyclonal antibody produced by immunizing rabbits with a synthetic peptide derived from human GAD65/GAD67 (#PA5–38102, 1–2000, Invitrogen); a purified mouse monoclonal antibody to gamma-aminobutyric acid receptor (GABA A R) subunit alpha-5 (GABA A R alpha-5) produced by immunizing mice with a fusion protein containing a sequence from the cytoplasmic domain (amino acids 368–419) of human GABA A R alpha-5 (Clone N415/24, 1–500, UC Davis/NIH NeuroMab Facility); a purified mouse monoclonal antibody to gamma-aminobutyric acid receptor subunit alpha-1 (GABA A R alpha-1) produced by immunizing mice with a fusion protein (amino acids 355–394) from mouse GABA A R alpha-1 (clone N95/35, 1–500, UC Davis/NIH NeuroMab Facility); a purified mouse monoclonal antibody to a synthetic peptide (amino acids 411–422) of human glial fibrillary acidic protein (GFAP) (#75–240, 1–10,000, UC Davis/NIH NeuroMab Facility); a purified rabbit polyclonal antibody produced by immunizing rabbits with a synthetic peptide derived from the sequence of human enolase-2 (neuron specific enolase) (AB#9536, 1–1000, Cell Signaling Technology); a monoclonal antibody to beta-actin produced by immunizing mice with a synthetic peptide corresponding to a sequences in the amino-terminal of human beta-actin (AB#3700, 1:10,000; Cell Signaling Technology); a monoclonal antibody to beta-actin produced by immunizing rabbits with a synthetic peptide corresponding to a sequences in the amino-terminal of human beta-actin (AB#4970, 1:5,000; Cell Signaling Technology).

Techniques: Activation Assay, Expressing, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cirsiliol targets tyrosine kinase 2 to inhibit esophageal squamous cell carcinoma growth in vitro and in vivo

doi: 10.1186/s13046-021-01903-z

Figure Lengend Snippet: Cirsiliol binds with TYK2 and inhibits the kinase activity. a Computational docking model between cirsiliol and TYK2. b The detailed binding site of cirsiliol on TYK2. c Upper panel: the binding ability of cirsiliol on endogenic TYK2 in vitro , obtained via pull down assay. Down panel: the binding ability of cirsiliol to recombinant TYK2 protein. d The change of affinity response intensity with the passage of time. -60 to 0 s was set as the time before inject cirsiliol solution; 0–120 s was set as contact time between cirsiliol and TYK2; 120–180 s was set as dissociation time. e The variation of response intensity with the increase of cirsiliol concentration. f Upper panel: cirsiliol binding ability with mutant TYK2. Down panel: the protein level of TYK2 in 293 T cell line. 1A (V981A), 1’A (P982A), 2A (double mutant), WT: wild type. g Kinase assay performed with cirsiliol and TYK2. h p-STAT3 inhibition analyzed by ImageJ in three independent assays ( n = 3, * p < 0.05, ** p < 0.01). Student’s unpaired t -test in ( h ). KD, dissociation constant

Article Snippet: Active TYK2 (Cat#T21-11G) and inactive STAT3 protein (Cat#S54-54BH) were bought from Signal Chem company (Richmond, BC, Canada).

Techniques: Activity Assay, Binding Assay, In Vitro, Pull Down Assay, Recombinant, Concentration Assay, Mutagenesis, Kinase Assay, Inhibition

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cirsiliol targets tyrosine kinase 2 to inhibit esophageal squamous cell carcinoma growth in vitro and in vivo

doi: 10.1186/s13046-021-01903-z

Figure Lengend Snippet: Cirsiliol inhibits ESCC cell proliferation and the inhibiting effect is attenuated when TYK2 knockdown. a MTT assay showed that cirsiliol suppresses KYSE140 (left panel) and KYSE450 (right panel) cell viability. b Colony forming assay. Upper panel: the representative clone pictures of KYSE450 and KYSE140, scale bar: 200 µm; lower panel: Statistical analysis of the colony ratios of both cell lines after cirsiliol treatment. c Cell cycle arrest at the G2/M phase after cirsiliol treatment. Left panel: KYSE140 cells; right panel: KYSE450 cells. d The proliferation inhibiting ability of cirsiliol change after TYK2 knockdown (three independent groups were set up in each cell line and the data was analyzed between vehicle and treatment in each group separately). Upper panel: KYSE140 cells; lower panel: KYSE450 cells. Scale bar: 200 µm. e Representative colony pictures after TYK2 silencing with or without cirsiliol treatment. f Analysis of the colony number. Cell colonies were analyzed using Image-Pro Plus v6.0. g Cell viability after rescue TYK2 protein level in KYSE140 and KYSE450 TYK2 knockdown cell lines. h The change of TYK2 protein level and phosphorylation STAT3 after rescue TYK2 in KYSE140 and KYSE450. Student’s unpaired t -test was used in ( a , b , c , f , g ) and ANOVA in ( d ). Asterisks (** p < 0.01, *** p < 0.001) indicate a significant change

Article Snippet: Active TYK2 (Cat#T21-11G) and inactive STAT3 protein (Cat#S54-54BH) were bought from Signal Chem company (Richmond, BC, Canada).

Techniques: Knockdown, MTT Assay, Phospho-proteomics

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cirsiliol targets tyrosine kinase 2 to inhibit esophageal squamous cell carcinoma growth in vitro and in vivo

doi: 10.1186/s13046-021-01903-z

Figure Lengend Snippet: Cirsiliol inhibits ESCC through TYK2-STAT3 signaling pathways. a Immunofluorescence staining of KYSE140 and KYSE450: cells were treated for 24 h, and then stained for p-STAT3 (Tyr705) and STAT3 (100× magnification). b The analysis of p-STAT3 fluorescence intensity in KYSE140 and KYSE450 cells. Analyzed by student’s unpaired t -test (* p < 0.05, ** p < 0.01). c The change of STAT3 dimer formation after treated by cirsiliol in KYSE140 and KYSE450 cells. d The Nucleus localization variation of STAT3 after treated with 20 μM cirsiliol in KYSE140 and KYSE450 cells. e The effects of cirsiliol on the TYK2-related signal pathway in KYSE140 and KYSE450 cells. IOD, Integrated Optical Density

Article Snippet: Active TYK2 (Cat#T21-11G) and inactive STAT3 protein (Cat#S54-54BH) were bought from Signal Chem company (Richmond, BC, Canada).

Techniques: Protein-Protein interactions, Immunofluorescence, Staining, Fluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cirsiliol targets tyrosine kinase 2 to inhibit esophageal squamous cell carcinoma growth in vitro and in vivo

doi: 10.1186/s13046-021-01903-z

Figure Lengend Snippet: Cirsiliol inhibits ESCC patient-derived xenograft (PDX) tumor growth in vivo. a The information of two PDX cases. b The protein levels of TYK2 in different PDX cases. c The change of average tumor volume in different group of LEG73 and LEG104 cases after cirsiliol treatment (* p < 0.05) and tumor images of different groups after sacrifice. d Tumor weight analysis in different groups of LEG73 and LEG104 cases after cirsiliol treatment and the tumor growth inhibition of cirsiliol compared with the average tumor weight of the vehicle group (* p < 0.05, ** p < 0.01). e Upper panel: Representative IHC images of LEG73 and LEG104 tumor tissue slices (100 × magnifications), tumor tissues were stained with p-STAT3 and anti-Ki67; lower panel: Statistical analysis of IHC positive staining of p-STAT3 and Ki67 in both LEG73 and LEG104 cases (* p < 0.05, ** p < 0.01, *** p < 0.001). f Schematic diagram of the signaling pathway of esophageal cancer proliferation inhibited by cirsiliol. Data for each treatment group were compared to the control group and statistical significance was analyzed by student’s unpaired t -test

Article Snippet: Active TYK2 (Cat#T21-11G) and inactive STAT3 protein (Cat#S54-54BH) were bought from Signal Chem company (Richmond, BC, Canada).

Techniques: Derivative Assay, In Vivo, Inhibition, Staining, Control