Z devd fmk

According to previously reported methods (69 (link)), neutrophils were purified from mouse blood samples using Ficoll-Paque (Ficoll-Paque Plus, 1.077 g/mL, GE Healthcare, Chicago, IL, USA) and dextran (Sigma-Aldrich) sedimentation (3% w/v) density gradient centrifugation and red blood cell lysis. A flow cytometer (Gallios, Beckman Coulter, Brea, CA, USA) and a fluorescent anti-citrullinated histone H3 (CitH3) antibody (Abcam, Cambridge, UK) were used to investigate the neutrophil expression of NETosis marker CitH3 after treatments of supernatants from platelets or platelets plus NDs. To prepare the platelet supernatants, inhibitors (Z-WEHD-FMK, 10 μM, R&D Systems; Z-DEVD-FMK, 10 μM, R&D Systems; OLT1177, 10 μM, Cayman Chemical; NAC 150 ng/mL, Sigma-Aldrich; MitoTEMPO, 1 μM, Sigma-Aldrich; P-selectin: rP-sel, 100 ng/mL R&D Systems; 30 min pretreatments before addition of ND) were used to block ND-induced platelet activation and cell death. After treatments with or without NDs and inhibitors, platelet supernatants were harvested by centrifugation (2.5 x 10⁴ g, 10 min; Benchtop Centrifuge, ThermoFisher Scientific) to remove platelets and NDs. Peptidyl arginine deiminase 4 (PAD4) inhibitor GSK484 (10μM, Sigma–Aldrich, St. Louis, MO, USA) was used to block neutrophil NETosis in vitro and in vivo as described (69 (link)).

Protein lysates of normal lung tissue, adenocarcinoma tissues, squamous cell carcinoma tissues and large cell carcinoma tissues were purchased from OriGene Technology (Rockville, MD) (Supplementary Table 1). SAHA from Cayman Chemical Company (Ann Arbor, MI) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich Co.) at 10 mM as a stock solution. The pan-caspase inhibitor (Z-VAD-FMK; benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone), caspase-3 inhibitor (Z-DEVD-FMK; benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone), caspase-8 inhibitor (Z-IETD-FMK; benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone) and caspase-9 inhibitor (Z-LEHD-FMK; benzyloxycarbonyl-Leu-Glu-His-Asp-fluoromethylketone) were obtained from R&D Systems, Inc. (Minneapolis, MN) and were dissolved in DMSO at 10 mM to serve as stock solutions. TNF-α, TRAIL and FasL were also obtained from R&D systems, Inc. and were dissolved in water at 10 mg/ml as a stock solution. NecroX-2 and Necrostatin-1 from Enzo Life Sciences (Plymouth Meeting, PA) were dissolved in DMSO at 1 mM and 50 mM as a stock solution, respectively. Cells were pretreated with each caspase inhibitor for 1 hour prior to SAHA treatment. DMSO (0.01%) was used as a control-vehicle and it did not affect cell growth and death.