A375 melanoma cells were seeded onto 10-cm dishes at a density of 1.5 × 106, transfected 24 h later with 10 μg of Apo plasmid using Lipofectamine 2000, and another 24 h later reseeded into 48-well plates at 5 × 104 cells per well. After another 24 h, cells were treated with apoptotic stimuli; then plates were imaged every 2 h using an Incucyte Zoom system (Sartorius) to quantify the number of fluorescent cells per field of view.
Separately, A375 melanoma cells were transiently transfected with the Apo plasmid, then plated 24 h later in coated dishes (Ibidi), and then treated another 24 h later with hTNFa (20 ng/ml) + cycloheximide (1 μg/ml) alone or with the pan-caspase inhibitor Q-VD-OPh (20 μm ) for 24 h. Images were captured by epifluorescence microscopy and transillumination.
Separately, A375 melanoma cells were transiently transfected with the Apo plasmid, then plated 24 h later in coated dishes (Ibidi), and then treated another 24 h later with hTNFa (20 ng/ml) + cycloheximide (1 μg/ml) alone or with the pan-caspase inhibitor Q-VD-OPh (20 μ