Caspase 8

For Western blotting, total protein extracts were obtained in cell lysis buffer containing 150 mM NaCl, 1 mM EDTA, 1% NP-40, 50 mM Tris (pH 8.0) as well as phosphatase and protease inhibitors. Protein concentrations were determined by BCA staining (#23225, Thermo Fisher Scientific, Hennigsdorf, Germany) as compared to BSA concentration standard. Following SDS polyacrylamide gel electrophoresis, proteins were blotted on nitrocellulose membranes, as described previously [55 (link)].
Several primary antibodies were derived from Cell Signaling Technology (Danvers, MA, USA): caspase-3 (9662, rabbit, 1:1000), caspase-8 (9746, mouse, 1:1000), caspase-9 (9502, rabbit, 1:1000), Mcl-1 (4572, rabbit, 1:1000), Bcl-w (2724, rabbit, 1:1000) and Bcl-2 (2872, rabbit, 1:1000). Other primary antibodies were derived from Santa Cruz Biotech (Dallas, TX, USA): Bcl-x_L (sc-8392, mouse, 1:1000) and β-actin (sc-47778, mouse, 1:1000). As secondary antibodies, peroxidase-labeled goat anti-rabbit and goat anti-mouse were used (Dako, Hamburg, Germany; 1:5000).

To analyze protein expression and processing, cells were lysed directly with 1x SDS/PAGE sample buffer. Secreted proteins were isolated from cell supernatants by centrifugation at 2000 rpm for 10 minutes to remove cellular debris, followed by precipitation using trichloroacetic acid (TCA) overnight. Precipitated protein was pelleted by spinning at 13,000 rpm for 15 minutes at 4°C, then washed with ice-cold acetone, centrifuged at 13,000 rpm again for 10 minutes, before finally being suspended in 1x SDS/PAGE sample buffer. Samples were heated at 100°C for 5 minutes and then separated by SDS/PAGE and transferred to PVDF membranes (Millipore). Membranes were then probed with primary antibodies specific for murine caspase-11 (#C1354; Sigma-Aldrich), caspase-3 (#9662; Cell Signaling), caspase-8 (#4798; Cell Signaling), gasdermin D (#G7422; Sigma-Aldrich), IL-1β (12242S; Cell Signaling), and β-actin (#4967; Cell Signaling). Membranes were then probed with secondary antibodies anti-rat IgG (7077S; Cell Signaling), anti-mouse IgG (7076S; Cell Signaling), or anti-rabbit IgG (7074S; Cell Signaling). ECL Western Blotting Substrate and SuperSignal West Femto Substrate (Thermo Scientific) were used.

After infection for 4 days as described above, HFFs were washed 3 times with 4 ^oC PBS, and harvested using 0.25% trypsin (GIBCO). Following 3 more washes with PBS, the cells were lysed for 10 minutes at 4 ^oC using RIPA Buffer (Sigma) supplemented with Roche cOmplete protease inhibitor cocktail (Merck), and the protein fraction was collected from the supernatants after centrifugation (13,000g, 4 ^oC, 10 minutes). The proteins were then subjected to immunoblotting and probed with the following antibodies: IL-1β (R&D Systems, MAB601, 1:1000 dilution), ASC (Santa Cruz, sc-271054, 1:100 dilution), caspase-1 (Adipogen, AG-20B, 1:500), caspase-4 (Cell Signaling, 44505, 1:1000), caspase-8 (Cell Signaling, 94965, 1:1000), gasdermin D (Cell Signaling, 964585, 1:1000), NLRP3 (R&D Systems, MAB7578, 1:100), and β-actin (Sigma, A2066, 1:2000). Uncropped images of immunoblots are provided as S9 Fig.

Caspase-9, caspase-8, caspase-3, cleaved-caspase-3, poly(ADP-ribose) polymerase (PARP), XIAP, cIAP-1, cIAP2, Bcl-2, Bcl-xL, and Bax were procured from Cell Signaling Technologies (Beverly, MA, USA) and the GAPDH antibody was procured from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, United States). Annexin V-FITC, propidium iodide staining solution, Hoechst33342Solution, BD Cytofix/Cytoperm Plus fixation and permeabilization solution kit(BD (Pharmingen San Jose, CA, USA). The Cell Counting Kit-8 (CCK-8) kit and N-acetyl cysteine (NAC) was obtained from Sigma-Aldrich (St. Louis, MO, United States). z-VAD-FMK was bought from Calbiochem (San Diego, CA, USA). CellROXGreen, MitoSOXRed, andThiolTracker Violet were purchased from Invitrogen (Waltham, MA, USA). Mitopotential kit was purchased from the EMD Millipore Corporation (Danvers, MA, USA).

Curcumin, CCK-8 kit, DMSO, and N-acetyl cysteine were purchased from Sigma Chemical Co (St. Louis, MO, United States) (Caspase-9, caspase-3,cleaved caspase-3,PARP,XIAP,p-Akt,Akt,GSK3,P-GSK3,FOXO1,P-FOXO1,GAPDH,cIap1,cIap2, Bcl-2, Bcl-xl, caspase 8, and cleaved caspase-8 antibodies were obtained from Cell Signaling Technologies (Beverly, MA, United States). Bax, p-H2AX, and cytochrome c antibodies and cisplatin were procured from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, United States). Annexin V fluorescein isothiocyanate (FITC), Propidium Iodide (PI), and p-H2AX (pS139) antibodies were purchased from BD Biosciences (San Jose, CA). z-VAD-FMK was obtained from Calbiochem (San Diego, CA, United States). CellROX Green was obtained from Invitrogen (MA, United States). Curcumin was dissolved in DMSO and further diluted in the cell culture media for the treatment of cells, so that the final concentration of DMSO in wells is 0.1% at the highest concentration of Curcumin used in the study. Viability assays showed that 0.1% DMSO is non-toxic to the cells (data not shown).

Human colon cancer cells HT-29 (Cat# 300215) and CaCo-2 (Cat # 300137) were purchased from CLS (cell lines service, Germany). Cells were cultured in DMEM supplemented with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin at 5% CO₂, 37°C and 95% humidity. 3-methyladenine (3-MA) and Z-VAD-FMK were obtained from sigma-Aldrich. Antibodies against target proteins used in this study are: caspase 8, caspase 7, LC3 and Beclin-1 (Cell Signaling, USA); cleaved caspase 3, Cyclin B1, H3 phospho-Ser10, γH2AX (Millipore), TNFα, p62/SQSTMI and cleaved PARP (Abcam), survivin and β-actin (Santa Cruz Biotechnology).

After removing the media, the cells were washed in ice-cold PBS and solubilized in lysis buffer (50 mM HEPES (pH 7.2), 120 mM NaCl, 1 mM EDTA, 0.1% NP-40, 10% (w/v) glycerol, protease inhibitor cocktail) for 30 min on ice. The cells were then centrifuged at 13 000 × g for 15 min, supernatants were recovered and protein concentration was determined using the Bradford reagent (Sigma-Aldrich). Aliquots containing 4 mg of protein were incubated with 15 μg of goat polyclonal anti-caspase 8 (sc-6136, Santa Cruz) antisera for 2 h at 4 °C, then incubated overnight with protein G agarose beads (rec. protein G sepharose 4B conjugate; #101241, Thermo Fisher Scientific) at 4 °C under rotary agitation. The pelleted proteins were solubilized in SDS sample buffer, boiled for 5 min, clarified by centrifugation and subjected to SDS-PAGE and immunoblot analysis using the following primary antibodies: caspase 8 (#9746; 1:1000, Cell Signaling Technology), RIP1, FADD (sc-6035, 1:500, Santa Cruz Biotechnology).