Fmk008

Cells were seeded in triplicate in a 6-well plate with 30 × 10⁴ cells per well and allowed to recover for 24 h before drug treatment. Cells were treated with caspase-8 inhibitor (Z-IETD-FMK, FMK007, R&D Systems), caspase-9 inhibitor (Z-LEHD-FMK, FMK008, R&D Systems) or a pan-caspase inhibitor (Z-VAD-FMK, ALX-260-020-M001, Enzo Life Science) at 40 μM, 40 μM or 50 μM, respectively, for 18 h. Cells were treated with either 10 nM vincristine (Cayman Chemical) and incubated for 72 h, 10 nM actinomycin D (Cayman Chemical) for 48 h, 50 μM etoposide (Acros Organics) for 24 h or 0.5 µg/ml TRAIL (PHC1634,Gibco) for 12 h. For multidrug treatment, combinations of etoposide (50 μM) and vincristine (10 nM), or etoposide (50 μM) and actinomycin D (10 nM) were added to the cells and incubated for 24 h. Cell viability was determined using trypan blue staining, and cell number was counted in three different wells on blinded samples. For DNA methyltransferase inhibition assay, RH30 cells were treated with either DMSO (vehicle) or 5-aza-2′deoxycytidine (Sigma-Aldrich) at 30 µM and 60 µM for 48 h prior to RNA isolation. Culture medium supplemented with fresh drug was changed every 24 h. All assays were performed at least twice to confirm results.

Took out the myocardial tissue and cut the tissue into small fragments. According to the radioimmunoprecipitation assay (RIPA) instructions (Beyotime, Shanghai, China), an appropriate amount of prepared RIPA lysis solution was added and lysed at 4°C for 30 minutes, centrifuge at 12,000 g at 4°C for 15 minutes, and the supernatant was saved. At the same time, the bicinchoninic acid (BCA) kit (R&D, Minneapolis, MN, USA) was used to detect protein concentration. Then, the activity of Caspase3 (AF835) and Caspase9 (FMK008) in myocardial tissue was determined according to the instructions of the kit method (R&D, Minneapolis, MN, USA).

The experiment was performed as previously described (1 (link), 5 (link)). Anti–caspase-8 is the Z-IETD- FMK (fluoromethyl ketone; R&D Systems, #FMK007), and anti–caspase-9 is Z-LEHD-FMK (#FMK 008). For both caspase inhibition experiment, 100 µM (dissolved in DMSO) of each was added in the cell culture for 4 days. The medium was changed every second day.