Figure 8

PD 81,723 significantly reduced the expression of p21, AKT, VEGFR-2, and eNOS in HUVECs. HUVECs at 60–70% confluency in 6-well plates were treated with 0.05% DMSO or 50 µM PD 81,723 in either complete endothelial growth media or endothelial growth media deficient in VEGF-A for 24 h. The cells were harvested and protein was extracted 24 h post treatment, with the experiment repeated in triplicate. (A) PD 81,723 treated cells expressed significantly (*** = P < 0.001 vs. complete media control) lower levels of p21, independent of the addition of VEGF-A in the growth media. (B) There was no statistically significant change in endogenous VEGF-A mean protein levels in PD 81,723 treated cells. (C) AKT was decreased in the PD 81,723 treated cells; with a statistically significant (* = P < 0.05 vs. complete media control) decrease in PD 81,723 treated cells in complete media. (D) p-AKT appeared unchanged from observing the blots, but when comparing the ratio of p-AKT/AKT there was an increase in PD 81,723 treated cells; with a statistically significant (* = P < 0.05 vs. complete media control) increase in PD 81,723 treated cells in VEGF-A deficient media. (E) VEGFR-2 and p-VEGFR-2 did not present any detectable bands in PD 81,723 treated cells. (F) The levels of eNOS, and p-eNOS were not enough to present detectable bands in PD 81,723 treated cells. The band intensities for p21, VEGF-A, and AKT were divided by those of the corresponding loading control (β-actin) and p-AKT was divided by the total AKT result. The data was normalized to the results of the 0.05% DMSO treated samples in complete endothelial growth media. The error bars represent the standard deviation from three experiments. The blots for VEGFR2, p-VEGFR2, eNOS, and p-eNOS showed a definitive near absence of protein in PD 81,723 treated cells which did not require mean intensity quantification. Original blots are presented in Supplementary Fig. S4.