STI-571 decreases apoptosis induced by LatB and RGDfV in HBMECs. HBMECs (2 × 105 cells/well) were seeded and allowed to spread on VN-coated/HD-BSA–blocked 6-well plates in 0.4% BSA/RPMI 1640. Cells were then treated for 16 hours (I), 24 hours (A, D-H), or 48 hours (B-C) with vehicle, LatB (0.5μM) or RGDfV (5 μg/mL). Where indicated, STI-571 (5μM) was included 30 minutes before LatB or RGDfV. (A) Whole-cell lysates were resolved on 10% SDS-PAGE and analyzed by Western blotting for PARP cleavage. GAPDH served as loading control. (B-C) Apoptosis was assessed by flow cytometry using the Apo-Direct kit, measuring FITC-dUTP and PI content. Percentage of apoptotic cells in the 2 top quadrants is indicated. (B) Mean ± SEM of 3 independent experiments performed in triplicate as in panel C. P < .001 between presence or absence of STI-571 for each pair. (C) One representative experiment (percentage of apoptotic cells in the 2 top quadrants) is indicated. (D-E) Loss of mitochondrial membrane potential (ΔΨm) was measured by flow cytometry using JC-1 mitochondrial probe. (D) Representative flow cytometry plots. The values indicated represent the percentage of cells with depolarized mitochondrial membrane. The bars in panel E represent mean ± SEM percentage of cells with depolarized mitochondrial membrane potential from 2 experiments performed in triplicate. P < .001 between LatB with or without STI-571. P = .044 for RGDfV with or without STI-571. (F) Whole-cell lysates were analyzed for caspase-8 activity using the ApoTarget caspase-8/FLICE colorimetric protease assay. Bars represent mean ± SEM. P < .001 between LatB treatment with or without STI-571, P < .001 between RGDfV treatment with or without STI-571 (n = 4 replicates, shown is 1 of 2 experiments with similar results). (G-H) Whole- cell lysates were resolved on 12.5% SDS-PAGE and analyzed by Western blotting for caspase-8 cleavage. GAPDH served as loading control. (H) Mean densitometry of the cleaved caspase-8 p43/p41 fragment relative to GAPDH in 3 separate experiments. (I) HBMECs were cultured in 3D collagen as described in “Methods.” Cells were stained with Hoechst and annexin V–FITC, and photographed (original magnification ×400).
