Fig. 4: ZNF185 knockdown increases endothelial permeability via RhoA.
From: ZNF185 prevents stress fiber formation through the inhibition of RhoA in endothelial cells
a Endothelial permeability changes in response to PKA activity in HUVECs. Permeability was determined by measuring the passage of FITC-dextran via confluent HUVECs monolayers. Thrombin was used as a positive control. Two-sided Student’s t test, *p < 0.05, **p < 0.01 (n = 3). b ZNF185 knockdown increases endothelial permeability in HUVECs. After knockdown of ZNF185 by Sh-ZNF185, 10 µM forskolin or 10 U/ml thrombin was administered to HUVECs for 1 h. Permeability was measured as in Fig. 4a. *p < 0.05 (n = 3). c RhoA is activated using ZNF185 knockdown. RhoA activity in HUVECs was measured using a rhotekin pull-down assay. Representative blots are shown. *p < 0.05 (n = 3). d Y-27632 improves ZNF185 knockdown-induced stress fiber formation in HUVECs. Y-27632 (10 µM) was added to HUVECs transduced with scrambled shRNA or ZNF185 shRNA for 1 h. Immunofluorescence staining of VE-cadherin (magenta) and actin (green). Representative images are shown (n = 3). Scale bars, 50 µm. Intensity of stress fibers at the yellow lines was quantified in Supplementary Fig. 7a. e Y-27632 attenuates endothelial hyperpermeability induced by ZNF185 knockdown. Y-27632 (10 µM) was added to HUVECs transduced with scrambled shRNA or ZNF185 shRNA for 1 h. Permeability was measured as in Fig. 4a. *p < 0.05 (n = 3). Data are presented as the mean ±  standard error. Ctrl control, Fsk forskolin, PKI PKI 14-22 amide, myristoylated, Thr thrombin.
