Figure 4
Stable shRNA knockdown of PLD2 in EA.hy926 endothelial cells decreases eNOS protein expression levels and NO production. (A,B) Stable PLD2 knockdown cells (shPLD2 cells) were generated using shRNA lentiviral particles. In parallel, control shRNA sequences were used to generate a control cell line (Scramble cells). PLD2 knockdown was confirmed by qRT-PCR (A) and western blotting as shown in the cropped gel and blot images, respectively (B). Bar, 5 μM. The region of the blot containing the PLD2 immunoreactive band was scanned using an Odyssey CLx imaging system. (C) Confocal microscopy of eNOS protein expression as detected by immunofluorescent staining in Scramble and shPLD2 cells. Representative image of 3 experiments. (D) Western blotting of eNOS in Scramble and shPLD2 cells, representative blot (n = 3 experiments). The regions of the Western blots (Suppl. Figure 1) containing the eNOS and actin immunoreactive bands were scanned and the relative abundance of the individual samples quantified using an Odyssey CLx imaging system. (E) Quantification of western blot with eNOS levels normalized to actin (n = 3 experiments). (F) Measurement of nitrate production using a Griess Reaction Kit to quantify eNOS activity with and without treatment with a small molecule PLD2 inhibitor (NFOT) at 10 μM for 24 hours (n = 7 experiments). Mean ± SEM; ***p ≤ 0.001; E, Student’s t-test; F, one-way ANOVA with Bonferroni’s Multiple Comparison Test.
