Fig 1: NKCC1 inhibition influences endothelial permeability. (A, top left panel) endothelial cells immunostained with anti-NKCC1 antibodies show homogenous protein expression in a primary culture of wildtype (WT) and Nhe6-knockout endothelial cells (KO). (A, middle panel) NKCC1 immunofluorescence signal quantification. Samples from five mice were pooled per phenotype and experiment; (n = 3); Scale bar, 50 μm. Data represent the mean (SD); (n = 3); **P < 0.01. (A, top right panel) In Nhe6-knockout cells (KO), NKCC1 expression was downregulated, compared to WT cells. Data represent the mean ± SD; (n = 3); *P < 0.05. (A, bottom left panel) Representative western blot image showing decreased NKCC1 protein presence in Nhe6-knockout EC cell lysate. (A, bottom right panel) western bolt signal quantification. Each endothelial cell culture was obtained from three mice per phenotype and experiment, and individual experiments performed three times (n = 3); Data represent the mean (SD); **P < 0.01. (B) Relative mRNA expression of the junctional genes after inhibiting NKCC1 with 30 μM bumetanide (bum) in WT endothelial cells (EC). Error bars represent the (SD); (n = 3); ****P < 0.0001. (C) After inhibiting NKCC1 in WT ECs with 30 μM bumetanide (30 μM), permeability increased compared to untreated control cells (CTRL). Data represent the mean (SD); (n = 3). *P < 0.05.