Z devd fmk

The CLP rats were injected with 150 ng/g ODN2088 (agonist of TLR9, InvivoGen, San Diego, CA, USA), 50 ng/g SB203580 (inhibitor of p38 MApK, Cell Signaling Technology, Beverly, MA, USA), 75 ng/g PD98059 (inhibitor of ERK, Cell Signaling Technology), 150 ng/g SP600125 (inhibitor of JNK, Cell Signaling Technology), 100 ng/g Z-DEVD-FMK (inhibitor of apoptosis, Selleck, Houston, TX, USA), 12.5 ng/g INF39 (inhibitor of pyroptosis, Selleck), 37.5 ng/g Nec-1s (inhibitor of necroptosis, Cell Signaling Technology), and 1% dimethyl sulfoxide (DMSO) (vehicle, Sigma-Aldrich, St. Louis, MO, USA).^{[22 (link)–28 (link)]}

The cytotoxicity assay was performed as reported previously.⁴² Briefly, the PMA-pretreated THP-1 cells were incubated with V13 or δVFH at different concentrations (15.625–1000 nM). In the case where a specific inhibitor was used, the cells were pretreated with the inhibitor for 1 h and then treated with V13 or δVFH as above. The inhibitors included MCC950 (50 μM) (PZ0280, Sigma), Nec-1s (50 μM) (S8641, Selleck), Z-DEVD-FMK (50 μM) (S7312, Selleck), Ac-YVAD-CMK (50 μM) (SML0429, Sigma), NSA (20 μM) (S8251, Selleck), and BAPTA-AM (50 μM) (S7534, Selleck) targeting NLRP3, RIPK1, caspase 3, caspase 1, GSDMD, and Ca²⁺, respectively. After treatment, the cells were observed with a microscope (Ti-S/L100, Nikon, Japan), and lactate dehydrogenase (LDH) release was measured with the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (G1780, Promega) according to the manufacturer’s instruction. Briefly, the supernatant of the cells was transferred to a 96-well plate. An equal volume of CytoTox 96 Reagent was added to each well. The plate was incubated for 30 min at room temperature. Stop Solution was then added to the plate, and absorbance at 490 nm was determined with a microplate reader (Synergy H1, BioTek, USA).