Caspase 8

Fenofibrate, palmitic acid, propidium iodide and RNase A were purchased from Sigma Chemical Company (St. Louis, MO, USA). Annexin V-FITC apoptosis detection kit was purchased from BioVision (Mountain view, CA, USA). Antibodies to various proteins were obtained from the following sources: β-Actin was from Sigma Chemical Company. Bax, caspase-3, Cdk1, Cdk2, Cdk4, cyclin B and p21 were purchased from BD Biosciences (San Diego, CA, USA). Cyclin D1, MLKL(pS358) and RIP3(pS227) were purchased from Abcam (Cambridge, MA, USA). ACOT8, Bcl-2, caspase-8, CPT1A, CPT2, cyclin A, cyclin E, FABP1, FASN, PPT1, RIP1 and RIP3 were from GeneTex Inc (Irvine, CA, USA). Caspase-9 and RIP1(pS166) were purchased from Cell Signaling Technology Inc (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and -mouse IgG were from Abcam.

Protein samples were separated by 8%, 10%, or 12% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and then transferred onto PVDF membranes (EMD Millipore, Burlington, MA, USA). After being blocked with 5% non-fat milk, the membranes were incubated with the following primary antibodies at 4°C overnight: α-tubulin (GeneTex, Irvine, CA, USA), collagen I (GeneTex), α-SMA (Proteintech, Chicago, IL, USA), TGF-β (Cell signaling technology, Danvers, MA, USA), TNF-α (GeneTex), NLRP3 (Proteintech), caspase-1 (Proteintech), IL-18 (Proteintech), IL-1β (Proteintech), caspase-3 (GeneTex), caspase-8 (GeneTex), caspase-9 (GeneTex), and BCL-2 (GeneTex). Membranes were then incubated with HRP-conjugated mouse anti-IgG (EMD Millipore) or HRP-conjugated rabbit anti-IgG (EMD Millipore) secondary antibodies for 1 h. Membranes were developed using ECL detection reagent (EMD Millipore). Relative protein levels were quantified using Image J (Version 1.46, National Institute of Health, Bethesda, MD, USA), and protein densitometry were expressed relative to that of α-tubulin.

SAHA was synthesized by Prof. J. P Liou in Taipei Medical University [24 (link)] and was dissolved in dimethylsulfoxide (DMSO) (Fisher Scientific, 67-68-5). SB203580, a specific p38 inhibitor, was purchased from Selleckchem (S1076) and dissolved in DMSO. The following commercial antibodies were used in Western blotting and Immunofluorescence: CD133 (Proteintech, 18470-1-AP), Bmi1 (GeneTex, GTX114008), Nanog (GeneTex, GTX100863), Oct3/4 (Santa Cruz, SC5279), Vimentin (GeneTex, GTX100619), Sox2 (GeneTex, GTX101507), tubulin (Proteintech, 66031-1-lg), PARP (Cell Signaling, 9542S), caspase-8 (GeneTex, GTX110723), caspase-9 (Cell Signaling, 9502S), caspase-3 (Cell Signaling, 9661S), cyclin B1 (Santa Cruz, SC245), CDK1 (Santa Cruz, SC8395), p21 (Cell Signaling, 2947S), PCNA (Cell Signaling, 2586S), Phospho-p38 (p-p38, T180/Y182) (Cell Signaling, 9211S), p38 (Cell Singling, 8690S), p53 (Cell Signaling, 2524S), Phospho-p53 (Ser15) (Cell Signaling, 9284S), Phospho-p53 (Ser33) (Cell Signaling, 2526S), and Alexa Fluor 594-conjugated goat anti-rabbit IgG polyclonal antibodies (Invitrogen, A-11037).