N acetyl asp glu val asp 7 amido 4 trifluoromethylcoumarin

Caspase-3/7 activity was either measured by luminescence using the Caspase-Glo 3/7 (Promega) according to the manufacturer’s protocol or by fluorescence. The caspase activity assay was performed as follows using the fluorescent substrate N-acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethylcoumarin (Sigma-Aldrich). The cells were lysed directly in the medium by adding 5× lysis buffer (250 mM Hepes, 25 mM CHAPS, and 25 mM DTT) and pipetting up and down. 30 μl of lysed cells was incubated with 30 μl of 2× assay buffer (40 mM Hepes, 200 mM NaCl, 2 mM EDTA, 0.2% CHAPS, 20% sucrose, and 20 mM DTT), and 50 μM final concentration of substrate was taken in black opaque OptiPlate-96 (PerkinElmer) and read at 400/505 at 37°C every 2 min for 10 min.

Cells were plated at 150,000 cells/well in 6-well plates. After attachment and equilibration, cells were treated, as indicated, with ERK inhibitor U0126 for 30’, then incubated for 24 hrs with or without the indicated stimuli as described in the Experimental Set-up. Cells were harvested and caspase-3 activity was measured using the fluorogenic substrate N-Acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethylcoumarin (Ac-DEVD-AFC, Sigma-Aldrich). After harvesting, cells were lysed in 10 mM Tris-HCl, 10 mM NaH2PO4/NaHPO4 (pH 7.5), 130 mM NaCl, 1% Triton-X-100, and 10 mM Na4P2O7 and then incubated with 20 mM Hepes (pH 7.5), 10% glycerol, 2 mM DTT, and 25 μg/ml Ac-DEVD-AMC at 37°C for 2 h. The release of AFC was analyzed by the BioTek Synergy H1 Microplate Reader (BioTek Instruments Inc.) using excitation/emission wavelengths 390/510 nm. Relative caspase-3 activity values of stimulated vs. untreated cells were calculated.