Z vad fmk

For the detection of MLKL phosphorylation, necroptosis was induced by treatment with PBZ complex (25 μg/ml poly(I:C) (Tocris, #4287); 1 μM BV6 (APExBIO, B4653); 20 μM z-VAD-fmk(APExBIO, A1902)) or TBZ complex (1000 units/ml, recombinant human TNF-alfa (TNF) (R&D Systems, 210-TA-020); 1 μM BV6; 20 μM z-VAD-fmk) for 4 h. Whole-cell lysates were collected and used for western blotting. For the detection of caspase 3 cleavage, apoptosis was induced by combined PB (25 μg/ml poly(I:C) and 1 μM BV6) or TB (1000 units/ml TNF and 1 μM BV6) treatment for various times. Whole-cell lysates were collected and western blotting was performed.

ESCs were seeded at a density of 2 × 10⁵ cells/mL in the indicated media type in a clear 96-well plate. Inhibitors were administered 24 hr after seeding at a concentration of 50 μM (Z-VAD-FMK, ApexBio; Necrostatin-1, Selleckchem). At the indicated timepoints, lactate dehydrogenase (LDH) activity was quantified in the supernatant using the Pierce LDH Cytotoxicity Assay Kit (Thermo Scientific) according to the manufacturer’s instructions. A_680nm values were first subtracted as background noise. Then, absorbance from an average of 3 media-only wells (reflecting background LDH activity) was subtracted from every sample’s A_490nm value. Data were normalized to wells that had been lysed completely using the provided lysis buffer to establish a benchmark for 100% cell death.

Human HT29, HEK293T and HeLa cells were cultured in DMEM (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 100 μg/ml penicillin and streptomycin at 37 °C in a humidified incubator containing 5% CO₂. The following reagents were used: Hsp90 inhibitor 17AAG (Selleckchem, Huston, TX, USA, S1141); z-VAD-fmk (ApexBio, Huston, TX, USA, A1902); Triton X-114 (Sigma-Aldrich, St. Louis, MO, USA); anti-HA (sc-805), anti-Myc (sc-40), anti-RIP3 (sc-374639) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Hsp90 (ab178854), anti-phospho-MLKL (ab187091) (Abcam, Cambridge, MA, USA); anti-MLKL (Merck Millipore, Billerica, MA, USA, MABC604); anti-Flag M2 (Sigma-Aldrich); anti-RIP1 (BD Pharmingen, Franklin Lakes, NJ, USA, 51-6559GR). HRP-conjugated goat anti-mouse and anti-rabbit were from Thermo Scientific, Waltham, MA, USA. The smac mimetic SM-164 was a kind gift of Dr. Shaomeng Wang (University of Michigan Comprehensive Cancer Center, Ann Arbor, MI, USA).

Akata (EBV⁺) cells (gifts from Diane Hayward, Johns Hopkins University) were grown in RPMI 1640 media supplemented with 10% FBS (Cat# 26140079, Thermo Fisher Scientific) in 5% CO₂ at 37°C [105 (link),106 (link)]. The P3HR-1 cell (ATCC, HTB-62) was purchased from ATCC. The EBV-transformed lymphoblast cell lines (LCL, GM11830) was purchased from the Coriell Institute for Medical Research (Camden, NJ). The P3HR-1 cell was grown in RPMI 1640 media supplemented with 10% FBS. The LCL cell was cultured in RPMI 1640 media supplemented with 15% FBS. 293T cells (a gift from Diane Hayward, Johns Hopkins University) were grown in DMEM media supplemented with 10% FBS. The pan-caspase inhibitor (Z-VAD-FMK, Cat# A1902) was purchased from ApexBio.

Coibamide A was re-isolated from material collected by hand using SCUBA from Coiba National Park, Panama, and apratoxin A was isolated from a laboratory culture of a Red Sea strain of Moorea producens [33 (link),60 (link)]. Rapamycin, thapsigargin, and tunicamycin were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Oligomycin A was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) and Z-VAD-fmk from ApexBio (Houston, TX, USA). All compounds were reconstituted in 100% cell culture grade DMSO and stored at −20 °C until the day of treatment. The final concentration of DMSO was 0.1% for all studies. General laboratory reagents were from Sigma-Aldrich Corp. or VWR International (Radnor, PA, USA). Primary and secondary antibodies were commercial sources and used according to recommended protocols. Codes for primary antibodies from Cell Signaling Technology, Inc. (Danvers, MA, USA) were as follows: LC3A/B (#4108), ATG5 (D5F5U; #12994) as conjugated ATG5-ATG12, CHOP (#5554), BiP/GRP78 (#3177), acetyl-CoA carboxylase (#3676), GAPDH (#5174), alpha-tubulin (#2125), beta-actin (#4970S), caspase-3 (#9662S), and PARP-1 (#9532). Anti-SQSTM1/p62 (#ab91526) was from Abcam (Cambridge, MA, USA) and a second anti-ATG5 (N-term; #AP1812a) antibody was from Abgent, San Diego, CA, USA.

IL-2/α-IL-2 complexes (IL-2c) were prepared as follows. About 1 ug recombinant mouse IL-2 (Peprotech) was mixed with 5 ug α-IL-2 (Bioxcell, clone JES6-1) and incubated at 37 °C for 30 min. Mice were injected intraperitoneally (i.p.) with IL-2c for 3 consecutive days and sacrificed for analysis 48 h later.
In the IL-2c treatment experiment, for blockade of cell death in vivo, 10 mg/kg Z-VAD-FMK (Apexbio Technology) and 5 mg/kg Necrostatin-1 (Abmole) or DMSO in PBS were injected i.p. daily for 5 days. In the IL-2c treatment experiment, for blockade of FASL in vivo, 400 ug IgG (Bioxcell) or α-FASL (Bioxcell) in PBS was injected i.p. on day −1, 1, and 3, and mice were sacrificed for analysis on day 5.
For IL-33 treatment in vivo, mice were i.p. injected with 500 ng recombinant murine IL-33 (BioLegend) or PBS for 4 consecutive days and sacrificed for analysis on day 5.