Etoposide

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.

Cells were treated with different concentrations of etoposide (Sigma Aldrich) diluted from an initial stock of 10mM in DMSO or with 100μM CK666 (Millipore) from a 100mM stock. Equivalent volumes of DMSO were used as controls. For RNA-seq experiments, the control condition consisted of regular media, while the treated condition consisted of media in which the etoposide powder was dissolved directly to 5μM. For JMY rescue experiments, to be able to detect cleaved caspase-3 in the JMY^KO cell line, etoposide was dissolved to 10μM. For RNAi experiments, cells were grown in 6-well plates for 24h, transfected with 40nM siRNAs (S2 Table) using RNAiMAX (Invitrogen), incubated in growth media for 24h, reseeded into 6-well plates and 24-well glass-bottom plates (MatTek), and incubated for an additional 24h. Cells cultured in 6-well plates were collected and processed for immunoblotting or RT-PCR, and cells cultured in 24-well plates were used in live fluorescence microscopy assays.

Where indicated, HEK293T were treated with 100 μM Etoposide (Invitrogen), 20 μM Z-VAD-FMK (Invitrogen), 50 μM Necrostatin-1 (Enzo Life Sciences) and DMSO (Sigma) served was administered as vehicle.