Abt 737

Pantoprazole sodium in powder (PANTOLOC) was purchased from Takeda GmbH (Germany). Bafilomycin A1 (Baf-A1, S1413), Hydroxychloroquine Sulfate (HCQ, S4430), Rapamycin (Rapa, S1039), Wortmannin (WM, S2758), and Nutlin-3a (S8059), were purchased from Selleck Chemicals (Houston, TX, USA). N-acetyl-L-cysteine (NAC, A7250), 2′,7′-Dichlorofluorescin diacetate (DCFHDA, D6883), L-Glutathione reduced (GSH, G4251), Thapsigargin (TG, T9033), and 2-Aminoethyl diphenylborinate (2-APB, D9754) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Torin 1 (HY-13003), Tunicamycin (Tu, HY-A0098), Cycloheximide (CHX, HY-12320), Bortezomib (borte, HY-10227), and MG-132 (HY-13259) were purchased from MedChem Express (Shanghai, China). LysoTracker Red DND-99 (L7528), and Fluo-4,AM (F23917), Rhod-2,AM (R1245MP), MitoSOX™ Red (M36008), MitoTracker™ Green (M7514), MitoTracker™ Red CMXRos (M7512), and JC-1 Mitochondrial Membrane Potential Dye (T3168) were purchased from Molecular Probes (USA). ABT-263 (Navitoclax) (A3007) and ABT-737 (A8193) were purchased from Apex Bio (Houston, TX, USA). Lipofectamine 3000 (L3000008) and Lipofectamine RNAiMAX (13778150) were purchased from Invitrogen.

Twenty to 40 gastrula stage embryos were grown on OP50 bacteria for 2 days at 20 °C; nematodes collected, washed, and exposed to either 0.1% DMSO (Sigma, USA) as solvent control, or 10 μM ABT-737 (ApexBio, Taiwan). Earlier exposure to ABT-737 resulted in larval arrest. Animals were incubated in the drug solution with shaking for 24 h, pipetted onto plates, and either scored for aggregation or imaged.

GSC were seeded in three wells of a 6-well plate at a density of 5 × 10⁴ cells per well. Cell number for each well was assessed using a haemocytometer 1, 3 and 6 days after seeding. A median was calculated for each time point. Cell count was normalised to day 1 (n = 3 independent repeats per cell line).
Cell death and cell confluence were determined using live-cell imaging in the IncuCyte Zoom (Sartorius). Therefore, 12 × 10³ GSC per well were seeded in 96-well plates and treated with the described chemotherapeutic agents in the presence of 30 nM SYTOX Green (Life Technologies). Plates were applied to the IncuCyte imager at 37 °C in a humidified 95% air/ 5% CO₂ incubator. Over a period of 48 h, every hour two images were taken per well. Images were presented in green phase contrast at 10 × magnification and quantified using IncuCyte imaging analysis software (Sartorius). The following drugs were used: ABT737 (ApexBio, A8193), S63845 (Chemgood, C-1370), Actinomycin D (Calbiochem, 114666), Temozolomide (Sigma, T2577), Cisplatin (Selleck, S1166), Paclitaxel (Sigma, T7191). Percentage cell death was calculated by normalising against maximal cell death following treatment with 1 µM Actinomycin D, 10 µM ABT737 and 1 µM S63845 (100% cell death control, verified by visual inspection of IncuCyte images) (n = 3 independent repeats per cell line).

HEK293FT, SVEC4-10, MEFs and U2OS cells were cultured in high glucose DMEM supplemented with 10% FBS (Gibco #10438026), 2 mM glutamine (Gibco #25030081) and 1 mM sodium pyruvate (Gibco #11360070). Cells were cultured in 21% O₂ and 5% CO₂ at 37 °C. MEF Tnf^−/−Hoip^+/+ and MEF Tnf^−/−Hoip^−/− cell lines have been described before (Peltzer et al, 2014 (link)). SVEC4-10 cells were purchased from ATCC. All cell lines were routinely tested for mycoplasma.
The following chemicals were used in this study: ABT-737 (APEXBIO #A8193), S63845 (Chemgood #C-1370), Q-VD-OPh (AdooQ Bioscience #A14915-25), Doxycycline hyclate (Sigma-Aldrich #D9891), erastin (Biotechne #5449/10), TAK-243 (MedChemExpress #HY-100487), MLN4924 (Selleck Chemical #S7109), MG-132 (Selleck Chemical #S2619), and raptinal (Millipore Sigma #SML1745), MitoTracker Green FM (Invitrogen #7514), PKmito DeepRed (SPIROCHROME #SC055), oligomycin (Sigma-Aldrich # O4876), antimycin A (Sigma-Aldrich #A8674), rotenone (Sigma-Aldrich #R8875), cyclosporin A (Sigma-Aldrich # 30024).

Where indicated, apoptosis was induced using 10 µM Actinomycin D (Merck # 114666-5MG) with or without 10 µM ABT-737 (ApexBio #A8193) in full medium. The addition of Actinomycin D alone as well as together with ABT-737 lead to the robust induction of apoptosis after 18 h. However, apoptosis onset is very heterogeneous and the addition of ABT-737 lead to a more homogeneous induction. 20 µM QVD (ApexBio #A1901) was added to prevent the cells’ detachment from the coverslip.