Journal: Frontiers in Cellular Neuroscience

Article Title: Frizzled-9 impairs acetylcholine receptor clustering in skeletal muscle cells

doi: 10.3389/fncel.2014.00110

Figure Lengend Snippet: Fzd9 is dynamically expressed in skeletal muscle tissue. (A) Total RNAs were extracted from E14.5 and E19.5 mouse hind limb muscles and further subjected to RT-PCR to detect the mRNA expression of Fzd9 (+). As a negative control, samples were processed in the absence of reverse transcriptase (−). GAPDH expression was used as a loading control gene. The gel is representative of two experiments performed by triplicate. (B) Proteins from mouse hind limb skeletal muscles obtained at different developmental stages (E14.5, E16.5, and E19.5) were fractionated by SDS-PAGE and immunoblotted for Fzd9 and β-actin ( upper panel ). Fzd9 is expressed at the protein level in all the analyzed developmental stages since the expected 56kDa band is detected. A representative gel shows that Fzd9 band intensity displays a progressive decrease from E14.5 to E19.5. Data represent the mean ± SD of Fzd9/β-actin ratio from three experiments, normalized to E14.5 ( lower panel ) ( * p < 0.05, ** p < 0.01, ANOVA using Bonferroni's post-hoc analysis). (C) Whole-mounted diaphragms from E17.5 and P0 were stained with anti Fzd9 antibody (green) together with αBTX to reveal the postsynaptic densities (blue). Fzd9 is abundant in the synaptic domain of embryonic NMJs, where it displays a punctate expression pattern. The insets ( lower panels ) show that some αBTX-positive regions were not labeled with anti Fzd9 antibodies. Pictures are representative of at least three experiments performed by triplicate.

Article Snippet: Antibodies used were goat anti mouse Fzd9 (R and D Systems) as well as mouse or rabbit anti human β-catenin (Santa Cruz Biotechnology).

Techniques: Muscles, Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control, Reverse Transcription, Control, SDS Page, Staining, Labeling

Journal: Frontiers in Cellular Neuroscience

Article Title: Frizzled-9 impairs acetylcholine receptor clustering in skeletal muscle cells

doi: 10.3389/fncel.2014.00110

Figure Lengend Snippet: Fzd9 impairs agrin-dependent AChR clustering in myotubes. (A) Fzd9 is expressed in the muscle cell line C2C12 throughout differentiation. C2C12 cells were cultured in vitro and differentiated for 0, 3, or 6 days (d0-d6). Total proteins were subjected to Western blot analyses. An expected 56 kDa band is gradually increased during C2C12 cells differentiation. α-tubulin expression was used as a loading control. (B) C2C12 myotubes differentiated for 6 days were analyzed by immunocytochemistry to detect Fzd9. Fzd9 is localized to the plasma membrane of the myotubes (green, upper panel ), similar to Glut1, which was used as a marker of plasma membrane (red, middle panel ). The merge image ( lower panel ) reveals the co-localization of Fzd9 and Glut1. (C) Differentiated C2C12 myotubes were subjected to a sequential fractionation procedure to isolate samples enriched in cytoplasm (cyt) or plasma membrane (mb) proteins. Western blot analyzes showed that α-tubulin is specifically detected in cytoplasmic fractions, whereas the vitamin C transporter SVCT2 was only present in membrane-enriched protein fractions ( left panel ). C2C12 myoblasts were transfected either with GFP or Fzd9 and differentiated. Sequential protein lysates were analyzed by Western blot. Both endogenous Fzd9 (GFP-transfected cells) or overexpressed Fzd9 (Fzd9-transfected cells) were found predominantly in the plasma membrane and were absent in the cytoplasm. As a loading control, β-actin is found only in the cytoplasm-enriched fraction ( right panel ). (D) Myoblasts transfected with plasmids coding for GFP (control) or Fzd9 were differentiated into myotubes and subsequently incubated with 200 pM neural agrin. αBTX staining allows the visualization of the AChRs (red). Automatized quantification of aggregates shows that Fzd9 overexpression induces a decrease in the number of AChR clusters per myotube, as well as a reduction in the total area and average size of AChR clusters, compared to controls. Data represent the mean ± s.e.m. ( n = 3 performed by triplicate; normalized to GFP-transfected myotubes). ( ** p < 0.01, *** p < 0.001 compared to GFP controls, t -test).

Article Snippet: Antibodies used were goat anti mouse Fzd9 (R and D Systems) as well as mouse or rabbit anti human β-catenin (Santa Cruz Biotechnology).

Techniques: Cell Culture, In Vitro, Western Blot, Expressing, Control, Immunocytochemistry, Clinical Proteomics, Membrane, Marker, Fractionation, Transfection, Incubation, Staining, Over Expression

Journal: Frontiers in Cellular Neuroscience

Article Title: Frizzled-9 impairs acetylcholine receptor clustering in skeletal muscle cells

doi: 10.3389/fncel.2014.00110

Figure Lengend Snippet: Down-regulation of Fzd9 increases agrin-dependent AChR clustering in myotubes. (A,B) The efficiency of shFzd9 was tested by its ability to impair overexpression/function of the Fzd9 construct. (B) HEK293 cells were transfected with Fzd9HA together with a control shRNA (pFUX) or shFzd9, followed by protein homogenization and Western blot. Whereas a 56 kDa band corresponding to Fzd9 is detected in the membrane-enriched fraction of the control condition, Fzd9 expression is drastically silenced in cells transfected with shFzd9. (B) The efficiency of the shFzd9 to affect the functionality of Fzd9HA was assessed by co-transfecting Fzd9HA and Wnt2 in the presence or absence of the shFzd9 plasmid in HEK293 cells. Activation of the TOPflash luciferase reporter gene was used as a readout of activation of the canonical Wnt pathway. These experiments were performed at least three times by triplicate ( ** p < 0.01, *** p < 0.001, t -test). (C) Myoblasts were transfected either with GFP or shFzd9 and differentiated for 5 days. Myotubes were treated with neural agrin and further stained with an anti-Fzd9 antibody (red), along with αBTX (blue) to detect AChR clusters and DAPI (yellow) to stain nuclei. shFz9-transfected myotubes display silenced Fzd9 expression and an apparent increase in the number of AChR clusters compared to GFP-expressing myotubes. (D) Myoblasts transfected either with GFP or shFzd9 and grown for 5 days were treated with neural agrin and further stained with αBTX to detect AChR clusters (red). Myotubes expressing the GFP protein present in the shFzd9 plasmid show a significant increase in the number of AChR clusters, an increase in the total area of AChR clusters, as well as on the average size of AChR clusters, when compared to control myotubes that only express GFP. Data represent the mean ± s.e.m. ( n = 3 performed by triplicate; normalized to GFP-transfected myotubes). ( * p < 0.05, *** p < 0.001 compared to GFP controls, t -test).

Article Snippet: Antibodies used were goat anti mouse Fzd9 (R and D Systems) as well as mouse or rabbit anti human β-catenin (Santa Cruz Biotechnology).

Techniques: Over Expression, Construct, Transfection, Control, shRNA, Homogenization, Western Blot, Membrane, Expressing, Plasmid Preparation, Activation Assay, Luciferase, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: Frizzled-9 impairs acetylcholine receptor clustering in skeletal muscle cells

doi: 10.3389/fncel.2014.00110

Figure Lengend Snippet: Fzd9 enhances β-catenin accumulation in myotubes. (A) GFP and Fzd9-transfected myotubes were immunostained with an anti β-catenin antibody (red). Fluorescence intensity of transfected myotubes was quantified using Metamorph. Quantification of the data ( right panel ) shows that the expression of Fzd9 induces a significant ~2-fold accumulation of β-catenin in the sarcoplasma, compared to control GFP-expressing myotubes. (B) Total protein samples from GFP- and Fzd9-transfected myotubes were separated by SDS-PAGE and immunoblotted with Fzd9 and β-catenin antibodies. Quantification of the Fzd9 or β-catenin against β-actin band intensity ratios shows that Fzd9-overexpressing myotubes display a ~2-fold increase in β-catenin cytosolic levels, which is equivalent to the ~2-fold increase observed for Fzd9 levels, compared to control myotubes ( right panel ). Data represent the mean ± s.e.m. ( n = 3 performed by triplicate; normalized to control GFP cells; *** p < 0.001, t -test, compared to the GFP group). (C) Whole-mounted diaphragms of E17.5 mice were immunostained to detect Fzd9 (green) and β-catenin (red). AChR aggregates were stained with αBTX (blue). β-catenin is associated to the sarcolemma of embryonic muscle fibers, including the membrane domains where AChR clusters and Fzd9 are localized. Pictures are representative of at least three experiments performed by triplicate.

Article Snippet: Antibodies used were goat anti mouse Fzd9 (R and D Systems) as well as mouse or rabbit anti human β-catenin (Santa Cruz Biotechnology).

Techniques: Transfection, Fluorescence, Expressing, Control, SDS Page, Staining, Membrane

Journal: Nature Communications

Article Title: Endothelial cell-derived stem cell factor promotes lipid accumulation through c-Kit-mediated increase of lipogenic enzymes in brown adipocytes

doi: 10.1038/s41467-023-38433-5

Figure Lengend Snippet: a , b Representative immunoblotting and comparisons of c-Kit, pAKT1 (Ser473), AKT1, pAKT2 (Ser474), AKT2, pERK1/2 (Thr202/Tyr204), ERK1/2, ACL, ACC, FASN, and SCD1 before and 10 min (10 m), 6 h, and 12 h after either bovine serum albumin (BSA, 100 ng/ml) or SCF (100 ng/ml) treatment in Control (transfected with empty lentiviral plasmid) or c-Kit over-expressed (transfected with c-Kit cDNA inserted lentiviral plasmid) cultured BAs. The cultured BAs were pre-incubated in high glucose DMEM containing 1% FBS for 12 h before and after the treatment. The same amount of protein loading in each lane is verified by immunoblotting of tubulin. Dots and bars indicate mean ± SD from n = 3/group from two independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001 versus BSA by two-tailed t-test. Protein sizes are indicated as kilodalton (kDa). c , d Representative immunoblotting and comparison of effect of indicated signaling inhibitor on the SCF-induced increased protein level of SCD1 in the c-Kit over-expressed and differentiated cultured BAs. The cultured BAs were pre-incubated in high glucose DMEM containing 1% FBS for 12 h before and after the treatment. For the inhibitor experiments, either Dynasore (20 µM), SU5402 (1 µM), wortmanin (30 nM), AKT1/2 inhibitor (25 nM), PD 0325901 (1 µM), ruxolitinib (10 µm) or Y27632 (10 mM) was treated to cultured cells for 1 hr, and then SCF (100 ng/ml) or VEGF (100 ng/ml) was added and incubated for 12 h. Each dot indicates a value from one experiment and n = 3/group from two independent experiments. Vertical bars indicate mean ± SD. * P < 0.05 versus SCF by one-way ANOVA test followed by Tukey’s post-hoc test. Protein sizes are indicated as kilodalton (kDa).

Article Snippet: For the overexpression of c-Kit , mouse c-Kit cDNA (#MR227469, OriGene) was inserted in FUGW lentiviral plasmid (#14883, Addgene) tagging with GFP, and the resulting plasmid was transfected into pre-adipocytes for 24 h. As a control, the empty FUGW lentiviral plasmid was transfected into pre-adipocytes in the same manner.

Techniques: Western Blot, Control, Transfection, Plasmid Preparation, Cell Culture, Incubation, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1101/2024.01.02.573660

Figure Lengend Snippet: (A) Schematic of CRISPR/Cas9-mediated PLXNB2 knockout (KO) with small guide (sg) RNA targeting second coding exon. (B) Western blots show Plexin-B2 expression in different SD2 GSCs, with β-actin as loading control. Note Plexin-B2 precursor at 240 kDa and mature form at 170 kDa. (C) IF images show Plexin-B2 expression in different SD2 GSCs, with Hoechst nuclear counterstain. (D) Left, schematic of atomic force microscopy (AFM) indentation method to probe cell stiffness by cantilever deflection. Middle, AFM indentation curves of different SD2 GSCs; right, box plots of cell stiffness, showing 25– 75% quartiles, median (line), and mean (plus sign). n= 6 cells per group. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (E) Left, depiction of membrane tension measurement with optical tweezers. Middle, force measurements during tether extrusion (shaded box). Right, quantifications of tether extrusion forces. n=5 cells per group. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (F) Left, schematic of FLIM of cell membranes labeled with Flipper-TR membrane dye, with low and high membrane tension associated with shorter and longer lifetimes, respectively. Middle top, representative FLIM images, with lifetime heatmap shown on right. Middle bottom, images show similar fluorescence intensities of Flipper-TR dye in WT and PB2 KO cells. Right top, violin plots show fluorescence lifetime from 3 images per group. Two-sided unpaired t-test. Right bottom, phasor plots of FLIM image data, with arrow indicating a shift to shorter lifetime values for PB2 KO cells. (G) Model of Plexin-B2 regulation of cortical contractility and membrane tension. Phalloidin staining show differences of F-actin network in WT and PB2 KO SD2 GSCs. DAPI for nuclear staining. Arrows point to stress fibers and spread-out contours of the WT GSCs.

Article Snippet: The lentiviral vector for Dox-inducible Plexin-B2 overexpression was generated by inserting human PLXNB2 cDNA into a Dox controlled expression vector (pLenti-CMVtight-PLXNB2 iOE; deposited as Addgene #176849) .

Techniques: CRISPR, Knock-Out, Western Blot, Expressing, Control, Microscopy, Membrane, Labeling, Fluorescence, Staining

Journal: bioRxiv

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1101/2024.01.02.573660

Figure Lengend Snippet: (A) Top, timeline for dextran uptake assay. Bottom, live-cell imaging of WT and PB2 KO SD2 GSCs labeled with SPY-Actin and exposed to dextran-Alexa488. Enlarged images of boxed areas are shown below. Quantification of the areas of dextran + clusters per cell are shown in box plots, with 25–75% quartiles, median (line), and mean (plus sign). n=85 cells for WT, n=44 cells for PB2 KO. Mann–Whitney–Wilcoxon test. (B) Top, live cell confocal plane images of WT and PB2 KO GSCs with side views of z-stacks showing intracellular localization of diffuse dextran-Alexa 488 signals in PB2 KO cells in addition to dextran endosome signals. In contrast, WT cells contained only dextran + endosomes. Bottom, histograms show fluorescence profiles showing bimodal distribution of dextran-Alexa 488 fluorescence intensities in PB2 KO GSCs (blue and brown arrows). n=177 cells for WT, n=161 cells for PB2 KO. Mann–Whitney–Wilcoxon test. (C, D) Left, schematic of myr-palm-GFP or -CFP attached to inner membrane leaflet. Right, live cell fluorescence imaging at 72 hr after transfection shows internalization of myr-palm-GFP or -CPF on endomembranes (arrow) in WT GSCs, in contrast to membrane retention of the probes (arrowhead) in PB2 KO GSCs. (E) Left, schematic of TauSTED super-resolution microscopy of GSCs labeled with MemGlow. Middle, TauSTED live-cell images show reduced endosomes (arrowheads) in PB2 KO cells compared to WT. Right, box plots show areas of MemGlow clusters in each cell. n=26 cells for WT, n=13 cells for PB2 KO. Two-sided unpaired t-test. (F) Working model of regulation of cortical and membrane tension by Plexin-B2, affecting endocytosis and membrane permeability in GSCs.

Article Snippet: The lentiviral vector for Dox-inducible Plexin-B2 overexpression was generated by inserting human PLXNB2 cDNA into a Dox controlled expression vector (pLenti-CMVtight-PLXNB2 iOE; deposited as Addgene #176849) .

Techniques: Live Cell Imaging, Labeling, MANN-WHITNEY, Fluorescence, Membrane, Imaging, Transfection, Super-Resolution Microscopy, Permeability

Journal: bioRxiv

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1101/2024.01.02.573660

Figure Lengend Snippet: (A) Left, schematic of PH(PLCδ1)-GFP PIP2 probe. Right, live-cell imaging at 72 hr post transfection reveals that the PH(PLCδ1)-GFP probes were largely internalized in WT GSCs (arrow), but retained on membrane of PB2 KO GSCs (arrowhead). (B) Left, still images of videography show accumulation of the PH(PLCδ1)-GFP probes (arrow) in front of the nucleus (NucSpot) of migrating WT SD2 GSCs in tunnels, more so in 3 than 8 µm tunnel, but not in PB2 KO cells. Dashed lines delineate cell boundary. Long arrow denotes direction of migration. Right, quantifications of the ratio of PH(PLCδ1)-GFP fluorescence intensity at front vs. rear of GSCs during passage. n=13-16 cells per condition. One-way ANOVA followed by Tukey’s multiple comparison test. Data represent mean ± SEM. (C) Left, schematic of R(+8)-pre-GFP probe for negative surface charge of inner plasma membrane. Right, live-cell imaging at 72 hr post-transfection shows internalization of the probes (arrow) in WT GSCs, in contrast to the predominant membrane localization in PB2 KO GSCs (arrowhead). (D) Left, still images of videography show accumulation of the R(+8)-pre-GFP probes (arrow) at front zone of WT GSCs when traversing the 3 µm tunnel, but not in PB2 KO cells. Right, bar graphs show the ratio of R-pre-GFP fluorescence intensity at rear vs. front of GSCs when passing through tunnels. n=22 cells for WT, n=27 cells for PB2 KO. Mann–Whitney–Wilcoxon test. Data represent mean ± SEM. (E) Diagram illustrating voltage sensitive FluoVolt membrane dye, with fluorescent intensity quenched by voltage-sensitive electron transfer from electron-rich donor mediated by “molecular wire” in plasma membrane. (F) Left, FluoVolt live-cell imaging shows reduced FluoVolt fluorescent intensity in cell membrane of Plexin-B2 KO cells, consistent with higher negative charges of inner membrane. Right, box plots of membrane FluoVolt intensity. n=25 cells for WT, n=27 cells for PB2 KO. Two-sided unpaired t-test. Data represent mean ± SEM. (G) Left, still images from videography show higher FluoVolt fluorescent signals at rear zone (arrowhead) of WT GSCs when traversing tunnels, more so in 3 than 8 µm tunnel, but not in PB2 KO cells. Migration direction is denoted by long arrow. Right, bar graphs show the ratio of FluoVolt intensity at rear vs. front during confined migration. n=15 cells per group. One-way ANOVA followed by Tukey’s multiple comparison test. Data represent mean ± SEM. (H) Live-cell images and quantifications show the effects of constitutive active (CA) RAP1B-V12 or dominant-negative (DN) RAP1B-N17 on FluoVolt intensity in WT or PB2 KO GSCs. n=25 cells per group. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (I) Left, still images capture calcium localization (Fluo4-AM fluorescence, arrowhead) at the rear of WT GSCs when traversing tunnels, more so in 3 than 8 µm tunnel, but not in PB2 KO cells. Migration direction is denoted by long arrow. Right, bar graphs showing Fluo4-AM intensity ratio at rear vs. front in GSC during passage through tunnels. n=15-16 cells. One-way ANOVA followed by Tukey’s multiple comparison test. Data represent mean ± SEM. (J) Left, still images from videography show that calcium chelator BAPTA-AM disrupted the pattern of high FluoVolt signals at the rear of WT GSCs (arrowhead) during confined migration. Right, bar graphs show FluoVolt intensity ratio at rear vs. front of GSCs when traversing tunnels. n=21 cells for WT, n=16 cells for PB2 KO. Two-sided unpaired t-test. Data represent mean ± SEM. (K) Model of Plexin-B2 signaling affecting membrane surface charge and electric field during polarized confined migration, with PIP2 enrichment at cell front and Ca 2+ at rear zone, leading to asymmetry of FluoVolt and R(+8)-pre-GFP.

Article Snippet: The lentiviral vector for Dox-inducible Plexin-B2 overexpression was generated by inserting human PLXNB2 cDNA into a Dox controlled expression vector (pLenti-CMVtight-PLXNB2 iOE; deposited as Addgene #176849) .

Techniques: Live Cell Imaging, Transfection, Membrane, Migration, Fluorescence, Comparison, Clinical Proteomics, MANN-WHITNEY, Dominant Negative Mutation

Journal: bioRxiv

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1101/2024.01.02.573660

Figure Lengend Snippet: (A) Structure model of the extracellular domain of human Plexin-B2 show the locations of lock1 and lock2 mutations predicted to form disulfide bridges that lock the ring structure. (B) Western blots show absence of mature Plexin-B2 (170 kDa) in PB2 KO GSC, and expression of lock mutants in PB2 KO SD2 and SD3 GSCs. β-actin serves as a loading control. (C) Still images from videography show passage of GSCs (nuclei visualized by NucSpot) through microchannels with PB2 wildtype rescue construct but not lock mutants, nor PB2 with deletion of extracellular domain (dECTO). Chevrons point to 3 µm constrictions. (D) Box plots show velocity through constrictions, stalling time at constrictions, and sum of forward and backward movements, with 25–75% quartiles, minimal and maximal values (whiskers), median (line), and mean (cross). For velocity and sum of movements: n=17-20 cells per condition. For stalling time at constriction: n=14-28 cells per condition. One-way ANOVA followed by Dunnett’s multiple comparisons test. (E) Still images from videography show F-actin assembly (SPY-actin, arrowhead) at cell rear and MemGlow + endosomes (arrow) at cell front of SD3 GSCs with Plexin-B2 WT rescue but not mutant rescues when traversing 3 µm constrictions (chevrons). (F) Bar graphs showing fluorescence intensity ratio of SPY-actin and MemGlow at rear vs. front of GSCs during confined migration. n=10-18 cells per condition. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Data represent mean ± SEM. (G) Model of Plexin-B2 signaling and mechano-electrical regulation of membrane tension and membrane surface charge during polarized confined migration. Regionalized enrichment of endocytosis/PIP2 at cell front and F-actin/Ca 2+ at rear zone lead to asymmetry of FluoVolt and R(+8)-pre-GFP membrane probes.

Article Snippet: The lentiviral vector for Dox-inducible Plexin-B2 overexpression was generated by inserting human PLXNB2 cDNA into a Dox controlled expression vector (pLenti-CMVtight-PLXNB2 iOE; deposited as Addgene #176849) .

Techniques: Western Blot, Expressing, Control, Construct, Mutagenesis, Fluorescence, Migration, Membrane

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