Z vad fmk

RSV and cyclohexamide (Sigma-Aldrich, St. Louis, MO, USA), BafA1 (LC Laboratories, Woburn, MA, USA), Q-VD-OPH (BioVision, Milpitas, CA, USA), LysoTracker Red DND-99 (Invitrogen, Carlsbad, CA, USA), H₂DCFDA (Invitrogen), Hoechst 33342 (Invitrogen), NSA (EMD Millipore, Billerica, MA, USA), TRAIL (Peprotech, Rocky Hill, NJ, USA), NBD-C6-GluCer and NBD-C6-Cer (Avanti Polar Lipids, Alabaster, AL, USA) were obtained from the indicated suppliers. TNF (Ybdy, Seoul, South Korea), pan-caspase inhibitor z-VAD.fmk and bivalent IAP antagonist BV6 (Wuxi Apptec, Shanghai, China) were kindly provided by Professor David Wallach, Weizmann Institute of Science, Rehovot, Israel and Brefeldin A (Calbiochem, San Diego, CA, USA) by Dr. Yifat Merbl, Weizmann Institute of Science, Israel. Unless specified, all other chemicals and reagents were purchased from Sigma-Aldrich.
A549 lung adenocarcinoma cells and human HT29 colorectal cancer cells were obtained from ATCC (Manassas, VA, USA) and verified to be mycoplasma free by periodical PCR testing. They were maintained in DMEM and McCoy’s 5A medium (Biological Industries, Cromwell, CT, USA), respectively. Both media were supplemented with 2 mM glutamine (Gibco, Saint Aubin, France), 100 U/ml penicillin and streptomycin (Gibco) and 10% FBS (Hyclone, Logan, UT, USA). Transient transfections of siRNA and DNA were performed using Lipofectamine2000 (Invitrogen) as per the manufacturer’s instructions. siRNA validation screen was performed in a 96-well format in reverse-transfection mode using DharmaFect1 transfection reagent (Dharmacon, Lafayette, CO, USA).

TNF was obtained from Ybdy (Seoul, South Korea). The bivalent IAP antagonist BV6^{22 (link)} and the caspase inhibitor z-VAD.fmk were purchased from WUXI APPTEC (Shanghai, China). BHA, NAC, bacterial LPS, Nec-1, PI, 4-hydroxytamoxifen (4OHT), and Triton X-114 were from Sigma-Aldrich (St. Louis, MO, USA).