Figure 5. Hyperresponsiveness of ISCLS BOECs is eNOS dependent.

(A) Representative immunoblot of relevant receptors or signaling proteins in BOEC cell lysates (n = 5 donors/group). (B) Quantification of relative protein expression. Data indicate the mean ± SEM. n = 6–9 donors/group. NS, by 2-way ANOVA with Šidák’s multiple comparisons. (C) Relative CDH5 or NOS3 expression in BOECs evaluated by quantitative PCR (qPCR) (normalized to Actb and/or GAPDH). Data indicate the mean ± SEM. n = 6–10 donors/group. NS, by Mann-Whitney U test. (D) Relative intracellular Ca²⁺ concentrations in control (blue) or ISCLS-derived (red) BOECs stimulated with VEGF (100 ng/mL). Data indicate the mean ± SEM. n = 2–3 donors/group analyzed in 4–5 independent experiments. (E) Representative immunoblot showing p-eNOS^Ser1177 and total eNOS levels in lysates from BOECs stimulated with VEGF and immunoprecipitated with anti-eNOS antibody. (F) Quantification of the p-eNOS/eNOS ratio. Data indicate the mean ± SEM of 3–5 donors/group analyzed in 5 independent experiments. *P = 0.01 and ***P = 0.0008, by 2-way ANOVA with Šidák’s multiple comparisons. (G and H) Representative immunoblot (G) and quantification (H) of eNOS/actin in eNOS siRNA–transfected BOECs. Data indicate the mean ± SEM of 5 independent experiments. ****P < 0.0001, by 1-sample Student’s t test. (I) Representative TER in control or eNOS siRNA–transfected BOECs left untreated (blue and green) or stimulated with VEGF (red and magenta). (J) Maximum decrease in VEGF-induced TER from t = 0 as a percentage of the control siRNA response. Data indicate the mean ± SEM. n = 4–5 donors/group analyzed in 3–5 independent experiments. *P = 0.04, by unpaired, 2-tailed Student’s t test.