LatB transiently disrupts spreading of HBMECs but does not induce cell detachment. Endothelial apoptosis induced by RGDfV does not require cell detachment. (A) HBMECs were seeded on VN or PLL, both blocked with HD-BSA, and incubated for 24 hours. Where indicated, RGDfV (5 μg/mL) was added after cells were spread for 4 hours. Cells were photographed (original magnification ×400) or harvested, and lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting for PARP cleavage. Vinculin served as loading control. (B) HBMECs (3000 cells/well) were allowed to adhere and spread for 2 hours on VN-coated/HD-BSA–blocked 8-well chamber slides. RGDfV (5 μg/mL) or LatB (0.5μM) was added in 0.4% BSA/RPMI 1640 for 0.5, 2, 6, or 24 hours. Cells were permeabilized and stained with phalloidin, counterstained with DAPI, and photographed (original magnification ×400). (C) HBMECs were seeded on VN-coated/HD-BSA–blocked 6-well plates in 0.4% BSA/RPMI 1640. LatB (0.2μM) or vehicle was then added for 1, 2, or 20 hours (overnight), and plates were photographed. Shown are representative cells (all cells underwent the transient shape change after LatB treatment; original magnification ×400). (D) HBMECs (10⁴ cells/well) were seeded on VN-coated/HD-BSA–blocked 48-well plates and allowed to adhere and spread for 2 hours before LatB (0.2μM) in 0.4% BSA/RPMI 1640 was added. After overnight incubation, nonadherent cells were washed off and the adherent cells were enumerated using methyl-thiazol-tetrazolium assay. Data are mean ± SD; n = 16 for each mean. P > .05.