Fig. 3: Combination treatment induces caspase-dependent apoptosis. BxPC-3, MIA PaCa-2 and PANC-1 cells were seeded in 96-well plates at a cell seeding density of 3×10⁴ cells/cm²

a, b Caspase-mediated cleavage of caspase-8 and PARP was assessed by western blotting in cells treated with 100 μΜ gemcitabine for 24 h and/or 100 ng/ml TRAIL for 4 h for BxPC-3 and MIA PaCa-2 cells, and 4 and 6 h for PANC-1 cells (n = 3). One representative experiment is shown. Lysates were prepared and equal amounts (15 μg total protein) were subjected to SDS–PAGE, transferred to PVDF membranes and then immunoblotted with antibodies directed against a PARP (top panel), caspase-8 (middle panel) or GAPDH (bottom panel). b Caspase-mediated cleavages of caspase-8, PARP and BID in the presence or absence of the pan-caspase inhibitor Z-VAD-FMK (10 μM) were assessed by western blotting in cells treated as described above. Membranes were immunoblotted with antibodies directed against caspase-8, PARP and BID. GAPDH was used as a loading control. c The inhibition of cell survival following combination treatment was assessed in the presence or absence of the pan-caspase inhibitor Z-VAD-FMK. PANC-1 cells were seeded in 96-well plates at a cell seeding density of 3×10⁴ cells/cm². Cells were treated with 100 μM gemcitabine for 24 h and/or 100 ng/ml TRAIL for 6 h in the presence or absence of 10 μM Z-VAD-FMK. Cell survival was assessed using the MTT assay. All experiments were repeated three times and data are provided as means ± SEM (one representative experiment is shown). P-values were calculated using Student’s t-test to determine the statistical significance of the difference between cells treated with 100 μM gemcitabine and those treated with both 100 μM gemcitabine and 100 ng/ml TRAIL (*P < 0.05, **P < 0.01, ***P < 0.001)