Journal: eLife

Article Title: Arpin deficiency increases actomyosin contractility and vascular permeability

doi: 10.7554/eLife.90692

Figure Lengend Snippet: ( A ) Representative Western blots for WASP family Verprolin homolog 2 (WAVE2), WASP, the Arp2/3 subunit ArpC5, and arpin in lysates of control (shCtrl) and arpin-depleted (shArpin) human umbilical vein endothelial cells (HUVEC). The graph shows the mean pixel intensities of the protein bands normalized to the respective control bands. All bands were normalized to γ-tubulin as loading control (n=3). ( B ) Paracellular flux of 150 kDa FITC-dextran across confluent control and arpin-depleted HUVEC monolayers cultured on transwell filters (0.4 μm pore size) untreated or treated with TNFα for 18 hr. Data are represented as relative permeability normalized to control HUVEC set to 1 (n=9 in three independent experiments). ( C ) Representative Western blots for vascular endothelial (VE)-Cadherin, β-catenin, vinculin, and arpin in lysates of control and arpin-depleted HUVEC. The graph shows the mean pixel intensities of the protein bands normalized to the respective control bands (set to 1). All bands were normalized to γ-tubulin as a loading control (n=3). ( D ) Representative Western blots for ZO-1, claudin-5, and arpin in lysates of control and arpin-depleted HUVEC. γ-Tubulin was used as a loading control. The graph shows the quantification of the relative pixel intensity of ZO-1 and claudin-5 bands normalized to the untreated control (set to 1) and γ-tubulin (n=3). ( E ) Immunostaining of VE-Cadherin and β-catenin in control and arpin-depleted HUVEC (40 X objective, scale bar = 20 μm; dashed boxes indicate magnified areas that highlight changes in junctional architecture; 3.3 digital zoom, scale bars = 5 μm). Representative images of four independent experiments are shown. Arrows depict linear and mature junctions in shctrl HUVEC and interrupted and gap junction formation and junction internalization in shArpin HUVEC. All data are represented as mean ± SEM; ns: non-significant; *p<0.05; ***p<0.001; ****p<0.0001; two-tailed student’s t-test. Figure 3—source data 1. Uncropped and labeled membranes for . Figure 3—source data 2. Raw unedited membranes for .

Article Snippet: 60,000 HUVECs were seeded on 6.5-mm-diameter 0.4 μm pore transwell filters Corning-Costar, Acton, MA, coated with 0.8% gelatin and cultivated for 72 hr.

Techniques: Western Blot, Control, Cell Culture, Pore Size, Permeability, Immunostaining, Two Tailed Test, Labeling

Journal: eLife

Article Title: Arpin deficiency increases actomyosin contractility and vascular permeability

doi: 10.7554/eLife.90692

Figure Lengend Snippet: ( A ) Immunostaining of vascular endothelial (VE)-Cadherin with F-actin staining using phalloidin in control and arpin-depleted HUVEC treated with 100 μM of the Arp2/3 inhibitor CK-666 or vehicle (40 X objective, scale bar = 20 μm). Representative images of three independent experiments are shown. ( B ) Total phalloidin pixel intensity quantification normalized to the average of control HUVEC. Nine images were analyzed in each group from three independent experiments. ( C ) Central actin fiber density quantification done as described in . 100 cells were analyzed in each group from three independent experiments. ( D ) Paracellular flux assays using confluent control and arpin-depleted HUVEC monolayers on 0.4 µm pore transwell filters treated with 100 μM CK-666 or vehicle (n=7–9 from three independent experiments). Data are normalized to control HUVEC treated with the vehicle set to 1. All data are represented as mean ± SEM; ns: non-significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; two-tailed student’s t-test.

Article Snippet: 60,000 HUVECs were seeded on 6.5-mm-diameter 0.4 μm pore transwell filters Corning-Costar, Acton, MA, coated with 0.8% gelatin and cultivated for 72 hr.

Techniques: Immunostaining, Staining, Control, Two Tailed Test

Journal: eLife

Article Title: Arpin deficiency increases actomyosin contractility and vascular permeability

doi: 10.7554/eLife.90692

Figure Lengend Snippet: ( A ) Representative immunostaining of vascular endothelial (VE)-Cadherin with F-actin staining using phalloidin in control and arpin-depleted HUVEC treated or not with 10 μM of the ROCK1/2 inhibitor Y27632 (40 X objective, scale bar = 20 μm). ( B ) Phalloidin pixel intensity quantification normalized to the average of control HUVEC. 14 images were analyzed in each group from three independent experiments. ( C ) Central actin fiber density quantification of at least 140 cells in each group from three independent experiments. ( D ) Paracellular flux assays using confluent control and arpin-depleted HUVEC monolayers on 0.4 µm pore transwell filters treated with 10 μM Y27632 or vehicle (n=10–12 from four independent experiments). ( E ) Representative immunostaining of VE-Cadherin with F-actin staining using phalloidin in control and arpin-depleted HUVEC treated with 10 μM of the ZIPK inhibitor HS38 or vehicle (40 x objective; scale bar = 20 μm). ( F ) Phalloidin pixel intensity quantification normalized to the average of control HUVEC treated with the vehicle. 13 images were analyzed in each group from three independent experiments. ( G ) Central actin fiber density quantification of at least 125 cells in each group from three independent experiments. ( H–I ) Paracellular flux assays using confluent control and arpin-depleted HUVEC monolayers on 0.4 µm pore transwell filters treated with ( H ) 10 μM HS38 or vehicle (n=9–12 in four independent experiments) or ( I ) treated or not with 15 ng/mL TNFα and with 10 μM HS38 or vehicle (7–8 in three independent experiments). All immunofluorescences are representative of three independent experiments. All data are represented as mean ± SEM; ns: non-significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; two-tailed student’s t-test.

Article Snippet: 60,000 HUVECs were seeded on 6.5-mm-diameter 0.4 μm pore transwell filters Corning-Costar, Acton, MA, coated with 0.8% gelatin and cultivated for 72 hr.

Techniques: Immunostaining, Staining, Control, Two Tailed Test