Journal: Journal of Neuroinflammation

Article Title: TSG-6 attenuates inflammation-induced brain injury via modulation of microglial polarization in SAH rats through the SOCS3/STAT3 pathway

doi: 10.1186/s12974-018-1279-1

Figure Lengend Snippet: The effects of rh-TSG-6 on phosphorylation of Stat3 and cellular location of p-STAT3. The phosphorylation of STAT3 was assessed by immunofluorescent double-labeling. Immunoreactivity for p-STAT3 is attenuated at 24 h in the SAH + rh-TSG-6 group, as compared with the SAH + vehicle group. TSG-6 deficiency increased the expression of p-STAT3 and more translocation from the cytosol to the nucleus occurs was observed. Scale bar = 20 μm

Article Snippet: The following primary antibodies were used for WB: mouse anti-TSG-6 (Santa Cruz Biotechnology; 1:800), rabbit anti-STAT3 (Cell Signaling Technology; 1:2000), rabbit anti-phosphorylated STAT3 at Tyr705 (Cell Signaling Technology; 1:1000), rabbit anti-SOCS3 (Abcam; 1:1000), mouse anti-CD163 (AbD Serotec; 1:500), rabbit anti-CD86 (ProteinTech; 1:600), rabbit anti-IL-6 (PeproTech; 1:800), rabbit anti-IL-10 (ProteinTech; 1:600), and rabbit anti-β-actin (Cell Signaling Technology; 1:1000).

Techniques: Phospho-proteomics, Labeling, Expressing, Translocation Assay

Journal: Journal of Neuroinflammation

Article Title: TSG-6 attenuates inflammation-induced brain injury via modulation of microglial polarization in SAH rats through the SOCS3/STAT3 pathway

doi: 10.1186/s12974-018-1279-1

Figure Lengend Snippet: Effects of rh-TSG-6 and TSG-6 siRNA treatments on SOCS3/STAT3 axis. a Effect of exogenous TSG-6 on STAT3 pathway expression was evaluated by western blot and the relative IL-6, IL-10, SOCS3, p-STAT3, and STAT3 densities were quantificationally evaluated. b The representative western blot analysis of STAT3 axis in SAH rats accepted intracerebroventricular injection of siRNA as indicated and quantification of the level of IL-6, IL-10, SOCS3, p-STAT3 and STAT3 after deficiency of TSG-6. Data are expressed as the mean ± SD from six rats. ** P < 0.01

Article Snippet: The following primary antibodies were used for WB: mouse anti-TSG-6 (Santa Cruz Biotechnology; 1:800), rabbit anti-STAT3 (Cell Signaling Technology; 1:2000), rabbit anti-phosphorylated STAT3 at Tyr705 (Cell Signaling Technology; 1:1000), rabbit anti-SOCS3 (Abcam; 1:1000), mouse anti-CD163 (AbD Serotec; 1:500), rabbit anti-CD86 (ProteinTech; 1:600), rabbit anti-IL-6 (PeproTech; 1:800), rabbit anti-IL-10 (ProteinTech; 1:600), and rabbit anti-β-actin (Cell Signaling Technology; 1:1000).

Techniques: Expressing, Western Blot, Injection

Journal: Biochemical pharmacology

Article Title: Lyn attenuates sepsis-associated acute kidney injury by inhibition of phospho-STAT3 and apoptosis.

doi: 10.1016/j.bcp.2023.115523

Figure Lengend Snippet: Fig. 6. Lyn knockout aggravates kidney injury by activating phosphorylation of STAT3. Wild-type mice (Lyn+/+) or mice lacking the Lyn gene (Lyn−/−) were subjected to sham surgery or CLP (A-B). Some mice were also pretreated with the STAT3 inhibitor Stattic at 3 h before CLP (C-G). (A) Immunohistochemical staining of p-STAT3 in the kidney tissues. (B) Immu noblotting of p-STAT3 protein expression and density analysis of p-STAT3 expression in the kidney tissues. (C) Representative images of H&E and PAS staining of kidney tissue sections (magnification, 400×; bar = 50 μm). (D) Tubular injury scores of H&E staining for kidney damage. (E) BUN and sCr levels. (F) IL-1β, IL- 6, and TNF-α concentrations in mice serum of different groups. (G) Western blot and density anal ysis of p-STAT3, Bcl-2, Cleaved Caspase-3 and Bax in the kidney tissues. Data are representative of three independent experiments. Bars are represented as mean ± SD (n = 5–6 per group, one-way ANOVA with Tukey-Kramer post-test). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Next, slices were blocked with 3% BSA at room temperature for 30 min and incubated with anti-F4/80 (Cat# ab90247, RRID:AB_10712189; Abcam, Cambridge, MA, USA), anti-phosphorylated Lyn (Cat# 2731, RRID:AB_2138262; Cell Signaling Technology, Danvers, MA, USA) and anti-phosphorylated STAT3 (Cat# 9145, RRID:AB_2491009; Cell Signaling Technology, Danvers, MA, USA) at 4 ◦C overnight.

Techniques: Knock-Out, Phospho-proteomics, Immunohistochemical staining, Staining, Expressing, Western Blot

Journal: Biochemical pharmacology

Article Title: Lyn attenuates sepsis-associated acute kidney injury by inhibition of phospho-STAT3 and apoptosis.

doi: 10.1016/j.bcp.2023.115523

Figure Lengend Snippet: Fig. 7. Lyn reduces apoptosis, inflammation and phosphorylation of STAT3 in LPS-treated BUMPT cells. (A) Western blot analysis of p-STAT3, Bcl-2 and Cleaved Caspase-3. Cells were transfected with Lyn siRNA (50 nM) or negative control for 24 h before stimulation with 100ug/ml LPS for 4 h (p-STAT3, STAT3) and 24 h (Bcl-2, Cleaved Caspase-3). (B) Density analysis of p-STAT3, Bcl-2 and Cleaved Caspase-3 (siRNA transfection). (C) The mRNA levels of inflammatory markers TNF-α, IL-6 and IL-1β in cell supernatant. Cells were transfected with Lyn siRNA (50 nM) or negative control for 24 h before stimula tion with 100ug/ml LPS for 24 h. (D) Western blot analysis of p-STAT3, Bcl-2 and Cleaved Caspase-3. Cells were transfected with Lyn plasmid or negative control for 24 h before stimulation with 100ug/ml LPS for 8 h (p-STAT3, STAT3) and 24 h (Bcl-2, Cleaved Caspase-3). (E) Density analysis of p-STAT3, Bcl-2 and Cleaved Caspase-3 (Lyn plasmid trans fection). Data are representative of three independent experiments. Bars are represented as mean ± SD (n = 3 per group, one-way ANOVA with Tukey-Kramer post-test). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Next, slices were blocked with 3% BSA at room temperature for 30 min and incubated with anti-F4/80 (Cat# ab90247, RRID:AB_10712189; Abcam, Cambridge, MA, USA), anti-phosphorylated Lyn (Cat# 2731, RRID:AB_2138262; Cell Signaling Technology, Danvers, MA, USA) and anti-phosphorylated STAT3 (Cat# 9145, RRID:AB_2491009; Cell Signaling Technology, Danvers, MA, USA) at 4 ◦C overnight.

Techniques: Phospho-proteomics, Western Blot, Transfection, Negative Control, Plasmid Preparation

Journal: Biochemical pharmacology

Article Title: Lyn attenuates sepsis-associated acute kidney injury by inhibition of phospho-STAT3 and apoptosis.

doi: 10.1016/j.bcp.2023.115523

Figure Lengend Snippet: Fig. 8. Lyn agonist MLR-1023 protects CLP-induced AKI by inhibiting STAT3 phosphorylation. (A) BUN and sCr levels. Wild-type mice were pretreated with MLR-1023 (10, 30, 50 mg/kg i.p.) or DMSO 2 h before sham surgery or CLP. After 16 h, blood samples were collected. (B) Representative images of H&E and PAS staining of kidney tissue sections (Magnification: 400x, scale bar: 50 μm). Wild-type mice were pre treated with MLR-1023 (50 mg/kg i.p.) or DMSO 2 h before sham surgery or CLP. After 16 h, kidney tissues and blood samples were collected. (C) Renal tubular injury score. (D) BUN and sCr levels. (E) Elisa assay of serum IL-1β, IL-6, and TNF-α. (F) Western blot anal ysis of p-STAT3, Bcl-2 and Cleaved Caspase-3 in renal tissues. (G) Density analysis of p-STAT3, p-Lyn, Bcl-2 and Cleaved Caspase-3. Data are representative of three independent experiments. Bars are represented as mean ± SD (n = 5–6 per group, one-way ANOVA with Tukey-Kramer post-test). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Next, slices were blocked with 3% BSA at room temperature for 30 min and incubated with anti-F4/80 (Cat# ab90247, RRID:AB_10712189; Abcam, Cambridge, MA, USA), anti-phosphorylated Lyn (Cat# 2731, RRID:AB_2138262; Cell Signaling Technology, Danvers, MA, USA) and anti-phosphorylated STAT3 (Cat# 9145, RRID:AB_2491009; Cell Signaling Technology, Danvers, MA, USA) at 4 ◦C overnight.

Techniques: Phospho-proteomics, Staining, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Oridonin Targets Multiple Drug-Resistant Tumor Cells as Determined by in Silico and in Vitro Analyses

doi: 10.3389/fphar.2018.00355

Figure Lengend Snippet: Molecular docking studies of oridonin and known inhibitors on proteins involved in EGFR signaling pathway. Proteins have been represented in new cartoon format with different colors, while oridonin was represented in yellow. (A) Docking poses in to the pharmacophore of Akt2 kinase domain (PDB code: 3E87 in green cartoon representation). GSK690693 was represented in blue. (B) Docking poses in to the pharmacophore of EGFR tyrosine kinase domain (PDB code: 1M17 in gray cartoon representation). Gefitinib was represented in green and erlotinib was represented in blue. (C) Docking poses in to the pharmacophore of mTOR (PDB code: 4JSP in orange representation). Sirolimus was represented in blue. (D) Docking poses in to the pharmacophore of STAT3 DNA-binding domain (homology model created by using the template PDB code: 1BG1 in pink cartoon representation). NSC74859 was represented in blue. (E) Docking poses in to the pharmacophore of VEGFR1 (PDB code: 3HNG in black cartoon representation). Axitinib was represented in blue.

Article Snippet: Briefly, 1 million cells per well were seeded in 12-well plate, next day treatment with oridonin was performed, total protein were extracted after 24 h. The following primary antibodies (Cell Signaling Technology, Frankfurt, Germany) were used: anti-rabbit EGFR, anti-rabbit phosphorylated EGFR (Tyr1068) (1:1000), anti-rabbit STAT3, anti-mouse phosphorylated STAT3 (Tyr705) (1:1000), anti-rabbit Akt, anti-rabbit phosphorylated Akt (Ser473) (1:1000), and anti-rabbit β-actin (1:2000).

Techniques: Binding Assay

Journal: Frontiers in Pharmacology

Article Title: Oridonin Targets Multiple Drug-Resistant Tumor Cells as Determined by in Silico and in Vitro Analyses

doi: 10.3389/fphar.2018.00355

Figure Lengend Snippet: Western blot analysis of oridonin on EGFR pathway proteins. The effects of oridonin on phosphorylation of ΔEGFR, STAT3, and Akt were evaluated. Bands were normalized to β-actin in order to obtain numerical values (mean ± SEM of three independent experiments). Total EGFR, STAT3, and Akt protein levels are also shown. A representative blot is shown and statistical analysis was done by paired Student’s t -test. ∗∗ p < 0.01, ∗ p < 0.05.

Article Snippet: Briefly, 1 million cells per well were seeded in 12-well plate, next day treatment with oridonin was performed, total protein were extracted after 24 h. The following primary antibodies (Cell Signaling Technology, Frankfurt, Germany) were used: anti-rabbit EGFR, anti-rabbit phosphorylated EGFR (Tyr1068) (1:1000), anti-rabbit STAT3, anti-mouse phosphorylated STAT3 (Tyr705) (1:1000), anti-rabbit Akt, anti-rabbit phosphorylated Akt (Ser473) (1:1000), and anti-rabbit β-actin (1:2000).

Techniques: Western Blot, Phospho-proteomics

Journal: Frontiers in Pharmacology

Article Title: Maresin 1 Mitigates Sepsis-Associated Acute Kidney Injury in Mice via Inhibition of the NF-κB/STAT3/MAPK Pathways

doi: 10.3389/fphar.2019.01323

Figure Lengend Snippet: MaR1 inhibits NF-κB/STAT3 pathway activation in the kidney of sepsis mice. The expressions of p-p65, p65 (A) and p-Stat3, Stat3 (B) in renal tissues of mice in each group were analyzed by Western blot. The p-p65 protein levels normalized by p65 (C) , p-p65 protein levels normalized by β-actin (D) , p-Stat3 protein levels normalized by Stat3 (E) . Data are shown as mean ± SD, n = 9. ** P 0.01 vs. sham group; # P 0.05, ## P 0.01 vs. CLP group; && P 0.01 vs LD-MaR1 group. MaR1, Maresin 1; NF-κB, nuclear factor-kappa B; STAT3, signal transducer and activator of transcriptor 3; CLP, cecal ligation and puncture; LD-MaR1, MaR1 low-dose group.

Article Snippet: The membranes were incubated with the following primary antibody at 4°C overnight: phosphorylation index (p-p65, p-Stat3, p-JNK, p-ERK, p-p38), non-phosphorylation index (p65, Stat3), and β-actin (Cell Signaling Technology, Danvers, MA, USA) at 1:1,000.

Techniques: Activation Assay, Western Blot, Ligation

Journal: Frontiers in Pharmacology

Article Title: Maresin 1 Mitigates Sepsis-Associated Acute Kidney Injury in Mice via Inhibition of the NF-κB/STAT3/MAPK Pathways

doi: 10.3389/fphar.2019.01323

Figure Lengend Snippet: MaR1 inhibits p65 nuclear translocation in the kidney of sepsis mice. Representative immunofluorescence staining of kidney tissue at 24 h after CLP in each group of mice, and white arrow indicates p65 into the nucleus, magnification 1,000×. MaR1, Maresin 1; CLP, cecal ligation and puncture.

Article Snippet: The membranes were incubated with the following primary antibody at 4°C overnight: phosphorylation index (p-p65, p-Stat3, p-JNK, p-ERK, p-p38), non-phosphorylation index (p65, Stat3), and β-actin (Cell Signaling Technology, Danvers, MA, USA) at 1:1,000.

Techniques: Translocation Assay, Immunofluorescence, Staining, Ligation

Journal: Frontiers in Pharmacology

Article Title: Maresin 1 Mitigates Sepsis-Associated Acute Kidney Injury in Mice via Inhibition of the NF-κB/STAT3/MAPK Pathways

doi: 10.3389/fphar.2019.01323

Figure Lengend Snippet: MaR1 inhibits MAPK pathway activation and p65 nuclear translocation in the kidney of sepsis mice. The expressions of p-JNK, p-ERK, and p-p38 in renal tissues of mice in each group were analyzed by Western blot (A) . The p-JNK (B) , p-ERK (C) , and p-p38 (D) protein levels normalized by β-actin. (E) Nuclear translocation of p65 count/field. Data are shown as mean ± SD, n = 9. ** P 0.01 vs. sham group; ## P 0.01 vs. CLP group; && P 0.01 vs. LD-MaR1 group. MaR1, Maresin 1; MAPK, mitogen-activated protein kinase; CLP, cecal ligation and puncture; LD-MaR1, MaR1 low-dose group.

Article Snippet: The membranes were incubated with the following primary antibody at 4°C overnight: phosphorylation index (p-p65, p-Stat3, p-JNK, p-ERK, p-p38), non-phosphorylation index (p65, Stat3), and β-actin (Cell Signaling Technology, Danvers, MA, USA) at 1:1,000.

Techniques: Activation Assay, Translocation Assay, Western Blot, Ligation

Journal: bioRxiv

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

doi: 10.1101/2020.10.27.358374

Figure Lengend Snippet: (A) Stimulation of HUVEC with 25ng/ml recombinant human VEGF-165 protein for 2 and 5 minutes induces p-VEGFR-2 (Y1175) and p-STAT3 (Y705) via immunoblotting. (B) VEGF (25 ng/ml) stimulation in HUVEC for 10 and 30 minutes promotes co-immunoprecipitation of STAT3 and VEGFR-2. (C) VEGF (25 ng/ml) stimulation in HUVEC for 10 and 30 minutes promotes co-immunoprecipitation of p-STAT3 (Y705) and p-VEGFR-2 (Y1175). (D) GST pull-down of VEGFR-2 with STAT3. Lysates of HUVEC stimulated with serum for 30 minutes 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 immunoblot. GST-STAT3 and GST were both detected using an anti-GST antibody. (E) VEGF stimulation for 2 minutes and 5 minutes promotes nuclear localization of STAT3. DAPI is blue. Scale bar, 20 µm. (F) Quantification of nuclear immunofluorescence staining intensity. Mean ±SEM, one-way ANOVA. *P<0.05, ****<0.0001. (A-E) Images are representative of multiple biological replicates.

Article Snippet: The samples were analyzed by immunoblotting using anti-phosphorylated STAT3 antibody (Y705; Cell signaling Technology) to validate JAK2-mediated kinase activity upon STAT3 protein at the Tyr705 position.

Techniques: Recombinant, Western Blot, Immunoprecipitation, Negative Control, Binding Assay, SDS Page, Immunofluorescence, Staining

Journal: bioRxiv

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

doi: 10.1101/2020.10.27.358374

Figure Lengend Snippet: (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 display no overt vascular defects relative to WT. Vascular system visualized by microangiography with 2000 kDa FITC-dextran. (C) Microangiography using 70 kDa Texas Red-dextran permeabilizing tracer (red) and 2000 kDa FITC-dextran ISV marker (green) was performed on 3 days old VEGF-induced, STAT3 +/+ (n=30) and VEGF-induced, STAT3 -/- (n=9) zebrafish. (D) Quantitative analysis of vascular permeability upon VEGF stimulation in wildtype (STAT3 +/+ ) and knockout (STAT3 -/- ) zebrafish. **P<0.01, unpaired t-test (B, C) Representative images shown were obtained using a Zeiss Apotome 2 microscope with a Fluar 5x, 0.25 NA lens at RT. ISV: inter-segmental vessel.

Article Snippet: The samples were analyzed by immunoblotting using anti-phosphorylated STAT3 antibody (Y705; Cell signaling Technology) to validate JAK2-mediated kinase activity upon STAT3 protein at the Tyr705 position.

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

Journal: bioRxiv

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

doi: 10.1101/2020.10.27.358374

Figure Lengend Snippet: (A) Images of footpads from WT and STAT3 ECKO (endothelial cell-specific STAT3 KO) mice following tail vein injection with 1% Evans blue and human recombinant VEGF-165 protein (2.5 µg/ml) or PBS vehicle being injected into the root of the footpad. (B) Quantitation of Evans blue leakage in Tie2-Cre negative; STAT3 flox/flox (WT) and Tie2-Cre positive; STAT3 flox/flox (STAT3 ECKO ) mice. n=8 mice per group. Each mouse was injected with PBS on right anterior and posterior footpads and VEGF on left anterior and posterior footpads. Multiple biological replicates were performed and depicted findings are representative. *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA followed by Dunnett’s test.

Article Snippet: The samples were analyzed by immunoblotting using anti-phosphorylated STAT3 antibody (Y705; Cell signaling Technology) to validate JAK2-mediated kinase activity upon STAT3 protein at the Tyr705 position.

Techniques: Injection, Recombinant, Quantitation Assay

Journal: bioRxiv

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

doi: 10.1101/2020.10.27.358374

Figure Lengend Snippet: (A) Human VEGF-165 recombinant protein (VEGF; 25 ng/ml) stimulation of HUVEC promotes ZO-1 (green) disorganization at endothelial cell junctions (top: DMSO vehicle control pretreatment for 1 hour prior to VEGF stimulation). ZO-1 organization is maintained upon pretreatment with 30 μM AQ for 4 hours (middle) or 10 μM PYR for 1 hour (bottom) prior to VEGF stimulation. VEGF-induced phosphorylation of STAT3 at Y705 (p-STAT3; red) was reduced upon AQ or PYR pretreatment. Nuclei: DAPI (blue). Insets: ZO-1 staining trace of 1 representative cell/field. (A’) Quantification of p-STAT3 (red). (B) Serum-starved HUVEC were pretreated with DMSO (vehicle control) for 1 hour, 30 µM AQ for 4 hours, or 10 µM PYR for 1 hour prior to VEGF (25 ng/ml) stimulation for 0, 2 or 5 minutes. Lysates were immunoblotted. Please see for densitometry analysis. (C) Serum-starved HPAEC were pretreated with 10 µM PYR for 1 hour prior to VEGF (25 ng/ml) stimulation for 0, 5 or 30 minutes. VEGF stimulation promotes disorganization of ZO-1 (green) at endothelial cell junctions. ZO-1 organization is maintained when HPAEC were pretreated with PYR. VEGF-induced p-STAT3 (red) was reduced upon PYR pretreatment. Nuclei were stained with DAPI (blue). Insets: trace of ZO-1 staining on 1 representative cell per field. (C’) Quantification of p-STAT3 (red). (D) VEGF (25 ng/ml) stimulation of HMVEC-L promotes ZO-1 (green) disorganization at endothelial cell junctions. ZO-1 organization is maintained upon pretreatment with 20 μM PYR for 6 hours prior to VEGF stimulation. VEGF-induced p-STAT3 (red) was reduced upon PYR pretreatment. Nuclei: DAPI (blue). Insets: ZO-1 staining trace of 1 representative cell/field. (D’) Quantification of p-STAT3 (red). ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, one-way ANOVA.

Article Snippet: The samples were analyzed by immunoblotting using anti-phosphorylated STAT3 antibody (Y705; Cell signaling Technology) to validate JAK2-mediated kinase activity upon STAT3 protein at the Tyr705 position.

Techniques: Recombinant, Control, Phospho-proteomics, Staining

Journal: bioRxiv

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

doi: 10.1101/2020.10.27.358374

Figure Lengend Snippet: A-B) Serum-starved HUVEC were cultured in standard medium or astrocyte conditioned medium (mixed 1:1 with standard medium) and stimulated with VEGF (25 ng/ml). A) Cells were lysed and immunoblotted using indicated antibodies. B) IF was performed using p-STAT3 (Y705; red) and ZO-1 (green) antibodies. VEGF stimulation promotes disorganization of ZO-1 at endothelial cell junctions (i.e. jagged appearance). Nuclei stained with DAPI (blue). Insets: trace of ZO-1 staining. C) Quantification of the p-STAT3 staining intensity. *P<0.05, ***P<0.001, ****P<0.0001, one-way ANOVA.

Article Snippet: The samples were analyzed by immunoblotting using anti-phosphorylated STAT3 antibody (Y705; Cell signaling Technology) to validate JAK2-mediated kinase activity upon STAT3 protein at the Tyr705 position.

Techniques: Cell Culture, Staining

Journal: bioRxiv

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

doi: 10.1101/2020.10.27.358374

Figure Lengend Snippet: A) Serum-starved HUVEC cultured in astrocyte conditioned medium (mixed 1:1 with standard medium) were pretreated with 20 µM PYR for 1 hour prior to VEGF (25 ng/ml) stimulation for 0, 2, or 5 minutes. B) Serum-starved HUVEC cultured in astrocyte conditioned medium (mixed 1:1 with standard medium) were pretreated with 30 µm AQ for 4 hours or 10 µm PYR for 1 hour prior to VEGF (25ng/ml) stimulation. VEGF stimulation promotes disorganization of ZO-1 (green) at endothelial cell junctions (i.e. jagged appearance). ZO-1 organization is maintained when HUVEC are pretreated with AQ or PYR (i.e. smooth appearance). Nuclei were stained with DAPI (blue). Insets: trace of ZO-1 staining on 1 cell. C) Quantification of the intensity of phosphorylated STAT3 protein at Y705. *P<0.05, ****P<0.0001, one-way ANOVA.

Article Snippet: The samples were analyzed by immunoblotting using anti-phosphorylated STAT3 antibody (Y705; Cell signaling Technology) to validate JAK2-mediated kinase activity upon STAT3 protein at the Tyr705 position.

Techniques: Cell Culture, Staining

Journal: bioRxiv

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

doi: 10.1101/2020.10.27.358374

Figure Lengend Snippet: A) Serum-starved HUVECs were pretreated with 10 µM C188-9 for 5 minutes prior to human VEGF-165 protein (25ng/ml) stimulation for 0, 2 and 5 minutes. VEGF stimulation promotes disorganization of ZO-1 (green) at endothelial cell junctions. ZO-1 organization is maintained when HUVEC were pretreated with C188-9. p-STAT3 (Y705; red) was reduced upon treatment with C188-9. Nuclei were stained with DAPI (blue). B) Depiction of selected ZO-1 staining to help visualize its organization or disorganization upon VEGF treatment in the absence of STAT3 inhibitor, C188-9. C) Serum-starved HPAEC were pretreated with 10 µM C188-9 for 5 minutes prior to human VEGF-165 protein (25ng/ml) stimulation for 0 and 5 minutes. After stimulation with VEGF protein for 5 minutes, the structure of tight junction marked with ZO-1 was disrupted (i.e. jagged-like ZO-1 green staining). ZO-1 organization is maintained when HPAEC were pretreated with C188-9. Nuclei were stained with DAPI (blue). D) Depiction of selected ZO-1 staining. (E) Mice were administered C188-9 or vehicle prior to tail vein injection with 1% Evans blue and VEGF (2.5 µg/ml) or PBS vehicle being injected into the root of the footpad. Quantitation of Evans blue leakage in C57BL/6 wildtype mice. n=5 mice per group. Each mouse was injected with PBS on right anterior and posterior footpads and VEGF on left anterior and posterior footpads. Multiple biological replicates were performed and depicted findings are representative. *P<0.05, paired t-test.

Article Snippet: The samples were analyzed by immunoblotting using anti-phosphorylated STAT3 antibody (Y705; Cell signaling Technology) to validate JAK2-mediated kinase activity upon STAT3 protein at the Tyr705 position.

Techniques: Staining, Injection, Quantitation Assay

Journal: bioRxiv

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

doi: 10.1101/2020.10.27.358374

Figure Lengend Snippet: Zebrafish were exposed to embryo water containing DMSO or 25 μM PYR for 3 days at starting 6 hours post-fertilization. Protein lysates were harvested from 3 days post-fertilization embryos by sonication in RIPA buffer after removing yolk sac and immunoblotting was performed using antibodies against zebrafish p-STAT3 (Y708) and cofilin.

Article Snippet: The samples were analyzed by immunoblotting using anti-phosphorylated STAT3 antibody (Y705; Cell signaling Technology) to validate JAK2-mediated kinase activity upon STAT3 protein at the Tyr705 position.

Techniques: Sonication, Western Blot

Journal: bioRxiv

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

doi: 10.1101/2020.10.27.358374

Figure Lengend Snippet: (A) To perform a STAT3 GST pull-down of VEGFR-2 and JAK2, lysates of HUVEC stimulated with serum for 30 minutes 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. (B) JAK2 phosphorylates STAT3 in vitro. In vitro kinase assay was performed using purified human STAT3 protein and kinase active JAK2 protein. (C) Images of footpads from C57BL/6 wildtype mice treated with vehicle or JAK2 inhibitor AG490. Following tail vein injection with 1% Evans blue, human VEGF-165 protein (2.5 μg/ml) or PBS vehicle were injected into the root of the footpad. After 30 minutes, the mice were euthanized and the footpads were excised. (D) Quantitation of Evans blue leakage in C57BL mice treated with vehicle or AG490. n=4 mice per group. Each mouse was injected with PBS on right anterior and posterior footpads and VEGF on left anterior and posterior footpads. **P<0.01, ***P<0.001, paired t-test.

Article Snippet: The samples were analyzed by immunoblotting using anti-phosphorylated STAT3 antibody (Y705; Cell signaling Technology) to validate JAK2-mediated kinase activity upon STAT3 protein at the Tyr705 position.

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

Journal: bioRxiv

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

doi: 10.1101/2020.10.27.358374

Figure Lengend Snippet: (A) The pGL3-ICAM1-WT plasmid (top) containing the human ICAM1 promoter with a STAT3 binding site located at −115 to −107bp. The pGL3-ICAM1-SDM plasmid (bottom) with mutation in the STAT3 binding site as indicated. (B) HUVEC were transiently transfected via electroporation with pGL3-ICAM1-WT (Firefly), pRL-SV40 (Renilla) plasmids and different amounts of constitutively active STAT3 plasmid (1 µg and 3 µg) using Neon transfection system. Firefly and Renilla luminescence was measured and plotted as ratio. Mean ±SEM, two-tailed unpaired t-test. n=12 technical replicates. *P<0.05, ****P<0.0001. (C) Dual-luciferase assays were performed in HUVEC 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 ±SEM, two-tailed unpaired t-test. n=9 technical replicates. **P<0.01, ****P<0.0001. (D) Human VEGF-165 protein (25ng/ml) stimulated HUVEC lysates were immunoblotted for ICAM1, p-STAT3 (Y705), and total STAT3. (B-D) Depicted data is representative of multiple biological replicates. SDM: Site-directed mutagenesis.

Article Snippet: The samples were analyzed by immunoblotting using anti-phosphorylated STAT3 antibody (Y705; Cell signaling Technology) to validate JAK2-mediated kinase activity upon STAT3 protein at the Tyr705 position.

Techniques: Plasmid Preparation, Binding Assay, Mutagenesis, Transfection, Electroporation, Two Tailed Test, Luciferase