c-Abl is required in apoptosis induced by LatB and RGDfV. HBMECs (2 × 105 cells/well) seeded overnight on VN-coated/HD-BSA–blocked 6-well plates in 0.4% BSA/RPMI 1640 were transfected with nonspecific nonsilencing negative control siRNA, or c-Abl siRNA (100pM) for 5 hours or were mock-transfected (Lipofectamine 2000 without siRNA). LatB (0.5μM) or RGDfV (5 μg/mL) was added 30 hours after transfection. M indicates mock control; NC, nonspecific nonsilencing negative siRNA control; si, c-Abl siRNA. (A-B) Cells were collected 40 hours after LatB (0.5μM) or RGDfV (5 μg/mL) treatment (70 hours after transfection), and apoptosis was assessed by flow cytometry using the Apo-Direct kit, measuring FITC-dUTP and PI content. Percentage of apoptotic cells in the top quadrants is indicated. (A) Representative experiment of 3 with similar results. (B) Mean ± SEM of the 3 experiments. P < .001 between c-Abl siRNA and each of the controls (mock and nonspecific siRNA) in the presence of LatB- or RGDfV-treated groups. (C) Whole-cell lysates were collected after 24 hours of LatB (0.5μM) or RGDfV (5 μg/mL) treatment (54 hours after transfection) and analyzed by Western blotting for PARP cleavage and c-Abl (K-12 antibody). GAPDH served as loading control. (D) Whole-cell lysates were collected 24 hours after LatB (0.5μM) treatment (54 hours after transfection) and analyzed for caspase-3 activity using the ApoTarget caspase-3/CPP32 colorimetric protease assay. Bars represent mean ± SEM. P = .029 between nonspecific nonsilencing negative siRNA control with or without LatB. P = .18 between c-Abl-siRNA with or without LatB (n = 5 for each condition).
