Z levd fmk

For time-point stimulations of epithelial cells and stromal fibroblasts, cells were plated at a density of 1.5 × 10⁵ cells/ml in a 24 well plate and left to rest for 24 h before stimulation. Following the 24 h rest period, media was removed and replaced with control media or media containing 2 μg/ml LPS (Enzo Life Sciences, Exeter, UK) for 24, 12, 6, or 3 h before addition of 10 μM nigericin for 1 h.
For inhibitor experiments, cells were plated at a density of 1.5 × 10⁵ cells/ml in a 24 well plate and left to rest for 24 h before treatment. Cells were then treated with increasing concentrations of MCC950 (1–100 nM) (a kind gift from Prof. Luke O'Neill), Z-VAD-FMK (1–100 nM) (Invivogen) or Z-LEVD-FMK (0.02–2 μM) (Enzo Life Sciences) for 1 h before the addition of LPS for 6 h followed by addition of nigericin for 1 h.
Following stimulation cells were either pelleted and lysed in RIPA buffer supplemented with protease inhibitors for protein analysis or lysed in TRIzol for mRNA extraction and stored at −80°C. Supernatants were harvested and stored at −20°C for further analysis.

Saikosaponin (SSa; Nacalai Tesque Inc., Kyoto, Japan) was dissolved in DMSO (Sigma Chemical Co., St. Louis, MO) to form a 10-mM stock solution and stored at –20°C until use. Propidium iodide (PI), RNase A, Hoechst 33342, cytochalasin-B, Giemsa stain, and 4’,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma. Caspase colorimetric assay kits were purchased from BioVision (Palo Alto, CA). Caspase-4 inhibitors, z-LEVD-fmk and Ac-LEVD-CHO, were obtained from Enzo Life Sciences (Plymouth Meeting, PA). The caspase-12 inhibitor, z-ATAD-fmk, was obtained from BioVision. z-DEVD-fmk (caspase-3 inhibitor), z-VDVAD-fmk (caspase-2 inhibitor), and z-IETD-fmk (caspase-8 inhibitor) were obtained from Calbiochem (La Jolla, CA). The annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit was purchased from BD Biosciences (Franklin Lakes, NJ, USA).

F. novicida strain Utah (U112) and its isogenic mutant lacking the whole FPI^{61 (link)} were grown in tryptic soy broth (Conda) supplemented with 0.1% (w/v) cysteine. U937 cells, hMDMs, and BMDMs were plated at a concentration of 3 to 5 × 10⁴ cells in 96-well plates or 3 to 5 × 10⁵ in 24-well plates. One day before infection, hMDMs were gently detached and plated in media without antibiotics supplemented with 100 ng.mL^-1 M-CSF. U937 cells were treated 24 h with PMA before overnight stimulation with 100 U.mL^-1 IFN-γ (Immunotools) followed by infection. Bacteria were added onto macrophages at the indicated multiplicity of infection (MOI). The plates were centrifuged for 15 min at 1000×g and incubated for 1 h at 37 °C. Next, cells were washed and fresh medium with 10 μg.ml^-1 gentamycin (ThermoFisher Scientific) was added before incubation for the indicated time. When indicated, hMDMs were pretreated for 30 min before infection with the caspase inhibitors z-YVAD-FMK or z-LEVD-FMK (Enzo LifeSciences) at the indicated concentrations, inhibitors were maintained during infection. When indicated, infected hMDMs were transfected at 2 h post-invasion with 0.1 to 0.5 µg of poly(dA:dT) for 3 h.