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: Constitutively active STAT3 plasmid EF.STAT3C.Ubc.GFP was Addgene plasmid #24983 ( http://n2t.net/addgene:24983 ; RRID: Addgene_24983; deposited by Dr Linzhao Cheng) ( ).

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: Constitutively active STAT3 plasmid EF.STAT3C.Ubc.GFP was Addgene plasmid #24983 ( http://n2t.net/addgene:24983 ; RRID: Addgene_24983; deposited by Dr Linzhao Cheng) ( ).

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: Constitutively active STAT3 plasmid EF.STAT3C.Ubc.GFP was Addgene plasmid #24983 ( http://n2t.net/addgene:24983 ; RRID: Addgene_24983; deposited by Dr Linzhao Cheng) ( ).

Techniques: Inhibition, Control, 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: Constitutively active STAT3 plasmid EF.STAT3C.Ubc.GFP was Addgene plasmid #24983 ( http://n2t.net/addgene:24983 ; RRID: Addgene_24983; deposited by Dr Linzhao Cheng) ( ).

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: Constitutively active STAT3 plasmid EF.STAT3C.Ubc.GFP was Addgene plasmid #24983 ( http://n2t.net/addgene:24983 ; RRID: Addgene_24983; deposited by Dr Linzhao Cheng) ( ).

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: Constitutively active STAT3 plasmid EF.STAT3C.Ubc.GFP was Addgene plasmid #24983 ( http://n2t.net/addgene:24983 ; RRID: Addgene_24983; deposited by Dr Linzhao Cheng) ( ).

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

Journal: Nature communications

Article Title: TRIM28 modulates nuclear receptor signaling to regulate uterine function.

doi: 10.1038/s41467-023-40395-7

Figure Lengend Snippet: Fig. 1 | PGR interacts with TRIM28 to regulate decidualization in HESCs. a PR interacting proteins in decidual HESCs by RIME. b Immunofluorescence (IF) of TRIM28 (magenta) and PR (green) in pre-decidual and decidual HESCs. c TRIM28 and PR proximity (red dots, arrow pointed) in siNT and siTRIM28 treated pre-decidual and decidual HESCs by Proximity Ligation Assay.

Article Snippet: GFP tagged TRIM28 plasmid was purchased from Addgene (Plasmid #6539776).

Techniques: Proximity Ligation Assay

Journal: Nature communications

Article Title: TRIM28 modulates nuclear receptor signaling to regulate uterine function.

doi: 10.1038/s41467-023-40395-7

Figure Lengend Snippet: Fig. 2 | TRIM28 knockdown disrupt PR signaling in HESCs. a Edu labeled cell proliferation, migrated cells in the transwell assay, and brightfield images of pre- decidual and decidual HESCs. Yellow line circles the cells. b Comparison of siPGR and siTRIM28 mediated transcriptomic changes in the hormone stimulated decid- ual HESCs. c The heatmap of PR and TRIM28 binding peaks in decidual HESCs by CUT&RUN. d The overlapped peaks of TRIM28 and PR, the associated common DEGs altered by TRIM28 and PGR knockdown in the decidual HESCs. Genome

Article Snippet: GFP tagged TRIM28 plasmid was purchased from Addgene (Plasmid #6539776).

Techniques: Knockdown, Labeling, Transwell Assay, Comparison, Binding Assay

Journal: Nature communications

Article Title: TRIM28 modulates nuclear receptor signaling to regulate uterine function.

doi: 10.1038/s41467-023-40395-7

Figure Lengend Snippet: Fig. 3 | TRIM28 deletion impaired decidualization and steroid signaling in mouse uterus. a The representative pictures and immunofluorescence of FOXO1, GJB2, and COX2 at D4.5 uterus. Red arrows pointed to the embryo implantation sites. b The representative uterine pictures and Immunohistochemistry (IHC) of KI67 and HAND2 of decidual uterus upon artificial decidualization. Blue arrow pointed to the decidual horn. c The Immunohistochemistry of proliferative marker KI67 and

Article Snippet: GFP tagged TRIM28 plasmid was purchased from Addgene (Plasmid #6539776).

Techniques: Immunohistochemistry, Marker

Journal: Nature communications

Article Title: TRIM28 modulates nuclear receptor signaling to regulate uterine function.

doi: 10.1038/s41467-023-40395-7

Figure Lengend Snippet: Fig. 5 | TRIM28 deletion inhibited ERα activity. a Immunofluorescence (IF) of PR and ERα. b The overlapped binding peaks of TRIM28 and ERα, the associated common DEGs and top altered pathways between TRIM28d/d mice and E2 treated ovariectomized wildtype mouse uterus. c The genome browser of ERα, TRIM28 and H3K27AC binding peaks at Pgr. Blue line is the Chromatin loops predicted by HiC.

Article Snippet: GFP tagged TRIM28 plasmid was purchased from Addgene (Plasmid #6539776).

Techniques: Activity Assay, Binding Assay

Journal: Nature communications

Article Title: TRIM28 modulates nuclear receptor signaling to regulate uterine function.

doi: 10.1038/s41467-023-40395-7

Figure Lengend Snippet: Fig. 7 | Abnormally accumulation of TRIM28 positive alone cells in the TRIM28d/d uterus. a Immunofluorescence (IF) of TRIM28 and PR in the TRIM28f/f

Article Snippet: GFP tagged TRIM28 plasmid was purchased from Addgene (Plasmid #6539776).

Techniques:

Journal: Nature communications

Article Title: TRIM28 modulates nuclear receptor signaling to regulate uterine function.

doi: 10.1038/s41467-023-40395-7

Figure Lengend Snippet: Fig. 8 | Epithelial TRIM28 deletion reduced Ihh expression and promoted progenitor cell accumulations at luminal epithelium. a The immuno- fluorescence (IF) of TRIM28 and PR, TRIM28 and Pgrcre-GFP at D2.5, D3.5, and D4.5 of the TRIM28f/f, eTRIM28d/d and TRIM28d/d mouse uterus. Arrow points to the TRIM28 positive cells in the luminal epithelium of the TRIM28d/d and eTRIM28d/d

Article Snippet: GFP tagged TRIM28 plasmid was purchased from Addgene (Plasmid #6539776).

Techniques: Expressing

Journal: eLife

Article Title: Generation of stress fibers through myosin-driven reorganization of the actin cortex

doi: 10.7554/eLife.60710

Figure Lengend Snippet:

Article Snippet: eGFP-NMIIA was a gift from Mathew Krummel (Addgene plasmid #38297; ).

Techniques: Recombinant, Plasmid Preparation, Transfection, Construct, Control, Concentration Assay, Software, Modification, Clinical Proteomics, Staining