Anti cleaved caspase 3 9661

Mouse monoclonal antibodies against HSV-1 gDDL6 (sc-21719), HSV-1 ICP0 (sc-53070), anti- human HVEM ANC3B7 (sc-65284) and anti-β-tubulin (sc-55529) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), mouse anti-human TNFα (MAB1021) and mouse anti-human IFNα (MAB411) from Chemicon/Millipore (Billerica, MA, USA), rabbit polyclonal antibodies anti-cleaved caspase 3 (#9661) and anti-pro-caspase 3 (#9662) from Cell Signaling Technology (Danvers, MA, USA), and mouse anti-actin monoclonal antibody from MP Biomedicals (Santa Ana, CA, USA). The secondary fluorescein isothiocyanate-conjugated and horseradish peroxidase-conjugated anti-mouse IgG antibodies were obtained from Chemicon/Millipore, the secondary goat anti-mouse IgG phycoerythrin (pe)-conjugated from Santa Cruz Biotechnology. RPMI medium, MEM eagle medium, L-glutamine, penicillin, streptomycin and fetal bovine serum were purchased from Lonza (Basel, Switzerland). All other chemicals and reagents, when not specifically indicated, were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Anti-BRD4 (ab244221; mouse Ab; 1:1000) and anti-BCL-2 (ab692; mouse Ab; 1:1000) Abs were purchased from Abcam (Cambridge, MA, USA). Anti-cleaved caspase-3 (9661; rabbit Ab; 1:1500), anti-cleaved PARP (5625; rabbit Ab; 1:1000), and β-actin (3700; mouse Ab; 1:4000) Abs were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). RPMI-1640 (Roswell Park Memorial Institute-1640) culture media and fetal bovine serum (FBS) were purchased from Gibco (Thermo Fisher Scientific, Inc., Carlsbad, CA, USA). OPT-0139 was obtained from JBKLAB Co., Ltd. (Seongnam, Republic of Korea), and cisplatin was purchased from Sigma Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO) for in vitro studies and 0.9% saline for animal studies.

Anti-HMGCS1 (sc-33829), anti-HMGCR (sc-27578), anti-RHOB (sc-180), and anti-PARP-1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti–cleaved caspase 3 (#9661) and anti–cleaved caspase 9 (#9501) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-Flag M2 (F3165) and anti–β-actin (A2228) antibodies were from Sigma-Aldrich (St. Louis, MO). Simvastatin (196-17801), Pravastatin sodium salt (162-19821), Lovastatin (125-04581), Compactin (033-17031), Fluvastatin sodium (069-05571), Atorvastatin calcium trihydrate (012-23901), and Pitavastatin calcium (163-24861) were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan).

Protoporphyrin IX (PpIX, P8293), zinc protoporphyria (ZnPP, 282820), tetrahydrofuran (THF, 401757), triethylamine (TEA, T0886), N-acetylcysteine (NAC, A7250), nicotinamide adenine dinucleotide phosphate (NADPH, 621706), hemin (51280), and 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, D8417) were purchased from Sigma (Shanghai, China). Isobutyl chloroformate (01086117) was purchased from Adamas (Shanghai, China). Ethylene diamine (2143) and chloroform (3084) were purchased from Damao (Tianjin, China). The methoxy (polyethylene glycol) succinimidyl carboxyl methyl ester (mPEG-NHS, PS1-H-1K), with an average molecular weight of 1000, was purchased from Ponsure (Shanghai, China). Zinc acetate (Z111245) was purchased from Aladdin (Shanghai, China). Cell Counting Kit-8 (CCK-8,C0039), 2′,7′-dichlorofluorescein diacetate (DCFH-DA, S0033S), and Mito-Tracker Green (C1048) were purchased from Beyotime (Shanghai, China). Anti-PCNA (60097) was purchased from Proteintech (Wuhan, China). Anti-cytochrome c (ab133504) was purchased from Abcam (Shanghai, China), and anti-cleaved caspase-3 (9661) was purchased from Cell Signaling Technology (Shanghai, China).

Cells were washed with PBS and then lysed with lysis buffer [1 M Tris, 2.5 M NaCl, 10% glycerol, 0.5 M glycerophosphate, 1% Tween 20, 0.5% NP-40 and a complete protease inhibitor tablet (Roche)] for 20 min on ice. The cell lysates were separated by 10 or 12% denaturing SDS-PAGE. The proteins were then transferred to nitrocellulose membranes (Whatman), blocked with PBS containing 5% milk and 0.05% Tween and incubated with specific primary antibodies overnight. After being washed with PBS containing 0.05% Tween, the blots were incubated with peroxidase-conjugated secondary antibodies labelled with horseradish peroxidase (HRP) (Sigma, Italy) and developed using ECL according to the manufacturer’s instructions. Rabbit polyclonal anti-GAPDH (ab 9485) and anti-procaspase-9 (ab 32068) were purchased from Abcam (Italy) and diluted 1:500. Rabbit anti-PARP (9542) and anti-p53 (Ser15-9286) were purchased from Cell Signaling Technology (Italy) and diluted 1:500. Anti-cleaved caspase-3 (9661), also purchased from Cell Signaling Technology, was diluted 1:1000. Rabbit anti-Bcl2 (ab 59348) and rabbit polyclonal anti-cyclin B1 (4138), purchased from Santa Cruz Biotechnology (Italy), were diluted 1:500. Rabbit anti-procaspase-8 (ab 49853) was purchased from Sigma and diluted 1:1000.

Biopsies were fixed in formalin and embedded in paraffin before sectioning and staining. Biopsies from patient III.2 were obtained with informed consent (see below) from the pathological archives at Hadassah Hebrew University Medical Center, Jerusalem, Israel. Healthy skin control biopsies were obtained from the Human Research Tissue Bank, Cambridge Biomedical Research Centre, Cambridge University Hospitals, UK. Deparaffinisation, haematoxylin and eosin (H&E) staining, cleaved caspase‐3 immunohistochemical staining (anti‐cleaved caspase‐3, #9661, RRID AB_2341188, Cell Signaling Technology) with haematoxylin counterstaining, and TUNEL assays (TdT In Situ Apoptosis Detection kit, #4810‐30‐K, R&D Systems) with methyl green counterstaining were performed by NDBbio Laboratories, LLC., Baltimore, MD. Stained slides were scanned (20× objective), and image files (SVS files) were processed using the QuPath software (https://qupath.github.io/; Bankhead et al, 2017).

Reagents were obtained from: MLN4924 (Cayman Chemical, Ann Arbor, MI), YHO-13351 and fumitremorgin C (FTC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI) (Sigma, St. Louis, MO, USA). Antibodies were purchased from the following sources: (anti-NEDD8 [ab81264], anti-γ-H2AX [ab81299], anti-NRF2 [ab137550], and anti-p27 [ab32034] (Abcam, Cambridge, MA, USA), anti-p21 [05-345] (EMD Millipore, Burlington, MA, USA), anti-cleaved caspase-3 [9661] (Cell Signaling Technology, Danvers, MA, USA), anti-β-tubulin [T7816] (Sigma), anti-ABCG2 [sc-58222] (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and sheep anti-mouse-horseradish peroxidase (HRP) and donkey anti-rabbit-HRP (Amersham, Pittsburgh, PA, USA).