Devd cho

Prior to experiments in FIECSs or cell lines, a dose-response of the different inhibitors was conducted to determine effective concentrations and toxicity. The pan-cysteine protease inhibitor, E64 or E64d (Bachem, Torrance, CA) was used at a final concentration of 10 µm in human cell lines and 1 µm in FIECs. The caspase-3/7 inhibitor, DEVD-CHO (BD Biosciences), was used at a final concentration of 10 µm in all cultures. The pan-caspase inhibitor, z-VAD-fmk (Enzo Life Sciences, Farmingdale, NY) was used at a final concentration of 10 µm in human cell lines and 1 µm in FIECs. The RIP1K inhibitor, necrostatin-1 (UBPBio, Aurora, CO), was used at a final concentration of 5 µm in FIECs and 10 µm in human cell lines. All inhibitors were dissolved in the diluent, in dimethyl sulfoxide (DMSO). Unless otherwise stated, inhibitors were added to DMEM plus 20% FBS at 37 °C in 5% CO₂ for 1 h prior to induction of death.

Human macrophage cell line THP-1 was obtained from MilliporeSigma (MilliporeSigma, USA). Macrophages were cultured in RPMI 1640-GlutaMAX™ medium supplemented with 10% FBS, 10 mM pyruvate carbonate, and 10 mM Na⁺ HEPES in humid air with 5% CO₂ at 37° C. Macrophages were subjected to 200 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, China) to differentiate into macrophages 24 hours prior to exosome treatment. Macrophages were cultured on non-tissue culture plates for 3 days and removed by PBS solution buffer (10 mM EDTA), and then seeded in tissue-culture plates (1×10⁵) per well for 24 hours prior to exosome or apoptotic vesicle treatment. 20 μg exosomes or apoptotic vesicles were used to treat macrophages for 24 hours and then lysed using lysis buffer (Sigma-Aldrich, China). For caspase-3 inhibition, 50 μM caspase-3 inhibitor DEVD-CHO (BD Pharmingen™, USA) was added to macrophages for 1 hour prior to exosome treatment.