Sm 164

The following reagents were used: zVAD-FMK (10 μM, Apex Bio), QVD (10 μM, Apex Bio), SM-164 (100 nM, gift from Shaomeng Wang), GSK’963 (referred as RIPK1i, 100 nM, GlaxoSmithKline), Nec-1 (10 μM Merck), Nec-1s (10 μM Merck), RO-3306 (9 μM, Merck), Thymidine (Sigma), BIM peptide (Kind gift of Tony Letai), FLAG-hTNF (10 ng/ml, Enzo), MG132 (1-20 μM, as indicated per cell line, see below, SIGMA), Recombinant Casp8 (1U Enzo). The following antibodies were used for western blotting: α-RIPK1 (1:1000, BD Biosciences), α-HA (1:1000, Roche), α-PLK1 (1:1000, Bethyl Laboratories), α-PLK1-pT210 (1:2000, AbCam), α-Cyclin B (1:1000, Cell Signaling), α-BUBR1 (1:1000, BD Bioscience), α-BUBR1-pT680 (1:1000, Kind gift of Geert J.P.L Kops), α-BUBR1-pT676 (1:1000, Kind gift of Erich A. Nigg), α-pH-H3 (1:2000, Millipore), α-Casp8 (for WB - post IP, 1:5000, MBL), α-Casp8 - for IP (7.5 μg/ml, C-20, Santa Cruz Biotechnology, α-FADD – for IP (7.5 μg/ml, Santa Cruz), α-FADD (1:1000 BD Biosciences), α-RIPK3 (1:1000 Proscience), α-RIPK3 (1:1000 Novus Biological) α-cFLIP (1:1000, Enzo), α-Myc (1:1000, clone 9E10,SIGMA), α-HSP90 (1:1000 Santa Cruz Biotechnology).

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).

The amount of ATP in treated cells was determined using CellTiter-Glo 2.0 kit (Promega;WI, USA). Two thousand cells per well were seeded in 96-well white plates and were treated with 1 ng/mL of murine TNF-α (Peprotech; NJ, USA), 10 µM of SM-164 (ApexBio; Texas, USA), 10 µM of QVD (R&D systems), 10 µM of Necrostatin-1 (Sigma-Aldrich) or milk-derived EVs (100 µg/mL). After 48 h, 75 μL of CellTiter-Glo was added to each well and the plates were incubated at room temperature for 10 min. Luminescence was recorded using a Spectromax M5 (Molecular Devices; CA, USA).

TNFα and TRAIL cytokines were purchased from Peprotech (Rocky Hill, NJ). Antibodies against cleaved caspase-3, phospho-inhibitor of NFκB (pIκB), p50, p65, Lamin B and FADD were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Pan-caspase inhibitor (Z-VAD-FMK) was purchased from Enzo Life Sciences (New York, NY). Ripa lysis buffer was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). Necrostatin-1, PS-1145 and actin antibody were purchased from Sigma Aldrich (St. Louis, MO). SM-164 was purchased from ApexBIO (Houston, TX). Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Lawrenceville, GA). Dulbecco's modified eagle medium (DMEM) cell culture medium and cell culture supplements were purchased from Invitrogen (Carlsbad, CA).