Strail apo2l

The human GCT cell line KGN (Riken BioResource Research Center, Ibaraki, Japan) was cultured in Dulbecco’s modified Eagle’s medium/nutrient mixture F12 (DMEM/F12, Sigma-Aldrich, St. Louis, MO, USA) with 5% fetal bovine serum (FBS) (Gibco, Waltham, MA, USA). Normal human fibroblast cells (F202 and N60, generously provided by Ted Tredget, University of Alberta) and normal human kidney cells (NKC, isolated from the proximal tubule, provided by Ron Moore) were cultured in DMEM/high glucose (Sigma-Aldrich) with 10% FBS. All culture media were supplemented with 2 mM L-glutamine, 100 U/mL penicillin, and 100 U/mL streptomycin) (Gibco). Cell line authentication was performed for KGN using short tandem repeat DNA profiling (Promega GenePrint 10 System, Madison, WI, USA) at the Genetic Analysis Facility at the Centre for Applied Genomics of The Hospital for Sick Children (Toronto, ON, Canada).
PAC-1 was generously provided by Paul Hergenrother (University of Illinois) and Hoechst 33,342 was generously provided by Linda Pilarski (University of Alberta). Other chemicals include recombinant human TRAIL (sTRAIL/Apo2L, Peprotech #310-04); carboplatin (Enzo Life Sciences #400-041); NucView 488 Caspase-3 substrate (Biotium #30029); and resazurin sodium salt (R7017), embelin (E1406), and gemcitabine hydrochloride (G6423-10) from Sigma-Aldrich.

Drugs used in this study were recombinant murine sTRAIL/Apo2L (Peprotech; NJ, USA), murine TNFα (Peprotech), Debio1143/AT-406 (Selleck Chemicals; Texas, USA), LCL161 (Selleck Chemicals), SM-164 (ApexBio; Texas, USA), Birinapant (ApexBio), BV6 (ApexBio), GDC-0512 (Selleck), doxorubicin (Sigma-Aldrich; MO, USA) and cisplatin (Sigma-Aldrich). The following antibodies were used: anti-cIAP1 (Enzo Life sciences; NY, USA), anti-cIAP2 (R&D systems; MN, USA), anti-XIAP (MBL International; MA, USA), anti-FADD (Abcam; Cambridge, UK), anti-TNFR1 (Abcam), anti-TRAIL-R2 (R&D systems), anti-caspase-3 (BD Biosciences; NJ, USA), anti-caspase-8 (Abcam), anti-RIPK1 (BD Biosciences), anti-RIPK3 (ProSci; CA, USA), anti-MLKL1 (Abcam), anti-phosphoS345-MLKL (Abcam), anti-CYLD (Thermo Fisher Scientific; Massachusetts, USA), anti-crmA (Santa Cruz Biotechnology; Texas, USA), rabbit anti-GFP (polyclonal in-house antibody), goat anti-rabbit FITC (Merck Millipore; MA, USA), rabbit anti-goat-FITC (Thermo Fisher Scientific), mouse anti-GAPDH (Merck Millipore), donkey anti-rabbit HRP conjugated antibody (GE Healthcare Life Sciences; NJ, USA), goat anti-rat HRP conjugated antibody (GE Healthcare Life Sciences), and rabbit anti-mouse-HRP conjugated antibody (Sigma).