Caspase 2

Cells were incubated in lysis buffer (1 % Triton X-100, 50 mM HEPES, 150 mM NaCl, 1 mM MgCl_2, 1 mM EGTA, 10 % glycerol, and protease inhibitor cocktail) for 1 h at 4 °C. Cell lysates were harvested by scraping and centrifuged at 14,000 rpm for 10 min at 4 °C. Normalized samples (using the BCA assay (Fisher Scientific, Pittsburgh, PA)) were run on SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes for western blotting. Bound antibody was detected using enhanced chemiluminescence reagent followed by exposure to film. Primary antibodies were used at the following dilutions and obtained from the following sources: Ago2 rabbit monoclonal (#2897, 1:500), caspase 2 mouse monoclonal (1:1000), caspase 3 rabbit monoclonal (1:1000), caspase 8 mouse monoclonal (1:1000), and caspase 9 mouse monoclonal (1:1000) (Initiator caspases sampler kit #12675), Drp1 rabbit monoclonal (#8570, 1:1000), GAPDH rabbit monoclonal (#2118, 1:5000), LC3B rabbit polyclonal (#2775, 1:1000), pan-actin rabbit polyclonal (#4968, 1:1000), PARP rabbit polyclonal (#9542, 1:1000), and PINK1 rabbit monoclonal (#6946, 1:500) antibodies were obtained from Cell Signaling Technology (Danvers, MA). ATG7 rabbit polyclonal antibody (PM039, 1:1000) was obtained from MBL International Corporation (Woburn, MA).

For detection of the expression of apoptosis relevant proteins, cells were grown to 70% confluence and lysed in RIPA buffer. The lysates (30 μg total protein per lane) were separated by SDS–PAGE, blotted onto PVDF-membrane and incubated with the appropriate primary antibodies followed by incubation with the HRP-conjugated secondary antibody (Amersham Pharmacia Biotech, Freiburg, Germany). Antigen visualization was performed by enhanced chemiluminescence (ECL-kit, Amersham Pharmacia Biotech). Primary antibodies used and their suppliers were as follows: anti-caspase-8 and anti-caspase-9 (StressGen Biotechnology, Canada), anti-caspase-3, anti-caspase-2, anti-caspase-7, anti-FADD, anti-Bcl-x_L, anti-Apaf-1, anti-BAD (Transduction Laboratories/BD, Heidelberg, Germany), anti-Bfl-1 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Bid, anti-c-FLIP, anti-XIAP, anti-c-IAP2 and anti-Survivin (R&D Systems, Wiesbaden, Germany), anti-Smac/DIABLO (Biocarta, Hamburg, Germany) and anti-Bax (PharMingen, Hamburg, Germany).

Female 6–8-week-old wildtype C57BL/6 mice from Jackson Laboratories (Sacramento, California) were used. MLKL knockout mice in the C57BL/6 background were obtained from Dr. Warren Alexander^{41 (link)}. Caspase-2 KO (007899) and Caspase-11 KO (024698) mice were obtained from The Jackson Laboratory (Sacramento, California). Oropharyngeal aspiration was performed on each mouse as previously described with an inoculum of 100 μl of a ~1.0 × 10⁶ CFU dose^{42 (link)}. Briefly, after being anesthetized (2% vaporized isoflurane) each mouse was hung upright by its incisors, the tongue gently pulled outward with blunt forceps, and the respective inoculum pipetted into the pharynx accompanied by coverage of the nares to achieved forced inhalation. All mice experiments were performed with protocols approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee and in agreement with the NIH Guide for the Care and Use of Laboratory Animals. Healthy baboons, with a median age 11 (Interquartile radio [IQR], 10–19) years old were intrabronchialy challenged with Spn (1 × 10⁹ CFU) using a bronchoscope^{43 ,44 (link)}. Between 5–7 days after infection during severe pneumonia animals were sacrificed and lung tissue was collected. All baboon experiments were performed using protocols approved by the Southwest National Primate Research Center Institutional Animal Care and Use Committee and in agreement with the NIH Guide for the Care and Use of Laboratory Animals.

Antibodies used for immunoblotting in this study were: rabbit anti-GSDMD antibody EPR19829 (Abcam, Cambridge, UK), rabbit anti-DFNA5/GSDME antibody EPR19859 (Abcam,), rabbit anti-γHexokinase #4959–9988 (Bio-Rad, Hercules, CA, USA), mouse anti-FLAG T0003 (Affinity Biosciences, Cincinnati, OH, USA), rabbit anti-caspase-1 (A-19) (Santa Cruz Biotechnology, Dallas, TX, USA), rat anti-caspase-2 #MAB3507 (Merck, Darmstadt, Germany), mouse anti-caspase-3 (clone 19) (BD BioSciences, Franklin Lakes, NJ, USA), rabbit anti-caspase-4 #4450S (Cell Signaling Technology, Danvers, MA, USA), mouse anti-caspase-5 (4F7) MBL International, Woburn, MA, USA), rabbit anti-caspase-6 (ARC0031 (Thermo Fisher Scientific, Waltham, MA USA), rabbit anti-caspase-7 (#9492) (Cell Signaling Technology), mouse anti-caspase-8 (1C12) (Cell Signaling Technology), rabbit anti-caspase-9 (#9502) (Cell Signaling Technology), rabbit anti-mouse IgG-HRP #A9044 (Sigma Aldrich, Burlington, MA, United States), donkey anti-rabbit-HRP #NA934 (Amersham, Little Chalfont, UK).
Q-VD-OPh was purchased from SMBiochemicals (Anaheim, CA, USA). Disulfiram was purchased from Selleckchem (Houston, TX, USA). TC13172 was purchased from MedChemExpress (Monmouth Junction, NJ, USA). All drugs were dissolved in dimethyl sulfoxide (DMSO) at 10 mM. The final concentration of the DMSO solvent in the assays was 1% regardless of drug concentration.

Unless otherwise specified, all reagents and chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO). Anti-human CD3 antibody (OKT3) was kindly provide by Dr. Gilliland of Janssen and used for stimulation of all primary PBMC. Fluorescently labeled antibodies for flow cytometry against: CD8, CD4, CD3, CD62L, annexin V, and their isotype controls (IgG1, IgG2A, IgG2B) were obtained from BD Biosciences (San Jose, CA), BioLegend (San Diego, CA) or eBioscience (San Diego, CA). APC-labeled CD62L antibodies and BV421-labeled annexin V antibodies used for confocal microscopy were obtained from BioLegend (San Diego, CA) and BD Biosciences (San Jose, CA), respectively. HIV-1 core antigen antibody (KC57-FITC/PE) for intracellular p24 staining was purchased from Beckman Coulter, Inc. (Miami, FL). Recombinant IL-2, also known as Teceleukin or Tecin (RO23-6019, Roche, MN) was obtained from National Cancer Institute, NIH. The Luciferase Assay System was purchased from Promega Corporation (Madison, WI). HIV-1 p24 ELISA kit was obtained from PerkinElmer Life Sciences, Inc. (Waltham, MA). Ficoll-Paque was purchased from GE Healthcare Life Sciences (Pittsburgh, PA). The Vybrant FAM poly-caspase assay from Molecular Probes-Thermofisher (Waltham, MA) was used for intracellular, active-caspase staining of pan-caspases. For individual caspase expression measurements, caspase-1, -2, -3/7, -6, -8, -9, and -10 fluorescent inhibitors of caspases (FLICA) were used to stain active-caspases in live cells from Bio-Rad (Hercules, CA). DMEM, RPMI1640, penicillin/streptomycin (Pen/Strep), fetal bovine serum (FBS), and HEPES were purchased from Invitrogen Corporation (Carlsbad, CA). The metalloproteinase inhibitor, BB-94 (Batimastat), TAPI-1 (TNFα protease inhibitor-1) and TAPI-2 were purchased from Santa Cruz Biotechnology. Caspase inhibitors Z-VDVAD-FMK (caspase 2), and Z-DEVD-FMK (caspases 3,6,7, and 10) were purchased from Santa Cruz Biotechnology, Z-DQMD-FMK (caspase 3), Z-IETD-FMK (caspase 8) and Z-LEHD-FMK (caspase 9) were purchased from Adooq, Ac-DEVD-CHO (caspase 3, 7) and Ac-LEVD-CHO (caspase 4) were purchased from Sigma-Aldrich, Belnacasan (caspase 1 and 4) and pan-caspase inhibitors Z-VAD-FMK and Q-VD-OPH were purchased from MedKoo Biosicences (Morrisville, NC) or SM Biochemicals LLC (Anaheim, CA).