Figure 6.
DAVLBH-induced VE-cadherin internalization partly depends on the inhibition of microtubule polymerization. (A) Paclitaxel suppressed DAVLBH-induced VE-cadherin internalization. HUVECs labeled with anti-BV9 were treated or not with DAVLBH for 4 h with or without paclitaxel pretreatment; then, the cells were washed with acid PBS and labeled with an Alexa Fluor secondary antibody. Representative images and image quantification are shown (n = 3). (B) Paclitaxel suppressed DAVLBH-induced VE-cadherin internalization. HUVECs pretreated with or without paclitaxel were treated with or without DAVLBH for 4 h, and the cells were lysed in RIPA with or without trypsin/EDTA to collect the intracellular and total VE-cadherin fractions, respectively. Quantification of intracellular VE-cadherin is shown (n = 3). (C) Paclitaxel inhibited the DAVLBH-induced phosphorylation of Src at Try416 and VE-cadherin at Y658. HUVECs pretreated with or without paclitaxel were treated with or without DAVLBH for 4 h. (D) Paclitaxel attenuated DAVLBH-mediated endothelial cell monolayer hyperpermeability. HUVEC monolayers pretreated with or without paclitaxel were treated with or without DAVLBH for 4 h. (E) Paclitaxel reversed the DAVLBH-mediated inhibition of internalized VE-cadherin recycling back to the cell membrane. HUVECs pretreated with or without paclitaxel were treated with or without DAVLBH for 4 h. Quantification of BV9/Rab11a colocalization is shown (n = 3). (F) Paclitaxel attenuated DAVLBH-induced lysosomal degradation of internalized VE-cadherin. HUVECs pretreated with or without paclitaxel were treated with or without DAVLBH for 4 h. Quantification of BV9/LAMP colocalization is shown (n = 3). Scale bar, 20 µm. The data are presented as mean ± SEM. ***P < 0.001 compared with the control group; #P < 0.05 and ###P < 0.001 compared with the DAVLBH group (one-way ANOVA with Tukey's post hoc comparison).