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Although c-FLIPS protein expression was undetectable before TRAIL administration in these two cell lines, its expression was drastically increased in cells that survived TRAIL treatment.
For combination treatment, cells were pretreated with ABT-737 for 1 h before TRAIL was added.
Briefly, the bottom 20% wells for cell survival before TRAIL treatment were removed from analysis.
To analyse the effect of cycloheximide on the protein level of FLIP-L, the cells were pretreated 1 h with cycloheximide before TRAIL or anti-CD95 was added.
To measure the caspase-8 activity triggered by the TRAIL receptors and not that produced from active caspase-3/7, we treated cells with a caspase-3/7 inhibitor, DEVD-CHO, 1 hour before TRAIL treatment.
Cells were pretreated 30 min with caspases inhibitors zVAD-fmk (100 μM, Bachem), zVDVAD-fmk (50 μM, Calbiochem), zIETD-fmk, zDEVD-fmk, and zLEHD-fmk (50 μM, R&D systems) before TRAIL or HDACIs treatments.
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