Nec 1

For inhibition experiments, cells were isolated, settled in RPMIcomp. supplemented with 10 mM HEPES and activated as described above. Inhibitors or ROS scavengers were added at least 20 min (in case of MitoTEMPO 1 h) before cell irradiation with 70 J/cm² of 375 nm or 214 J/cm² of 470 nm, at 37°C. For an additional control experiment, Trolox and catalase/SOD were added separately after irradiation. The cells were then incubated for an additional 3 h without an additional washing step in the presence of the inhibitors to allow for NET formation and fixed by 2% PFA. Pure medium or 100 nM PMA without irradiation were used as negative and positive controls, respectively. The following inhibitors and ROS scavengers were used in this study: GW-311616A hydrochloride (iNE, Axon Medchem) at 5 μM, 4-aminobenzoic acid hydrazide (4-ABAH, Cayman chemicals) at 100 μM, z-VAD-FMK (Promega) at 20 μM, necrostatin-1 (Nec-1, Enzo) at 50 μM, Y-27632-dihydrochloride (Abcam) at 20 μM, Cl-amidine (Merck Millipore) at 200 μM, MitoTEMPO (Sigma-Aldrich) at 5 μM, diphenyleneiodonium chloride (DPI, Sigma-Aldrich) at 1 μM, Trolox (Sigma-Aldrich) at 50 μM, PEG-catalase at 2,000 U/ml (Sigma-Aldrich), and a mixture of catalase (filtered, Worthington) and superoxide dismutase (SOD, Sigma Aldrich/Merck) at 2,000 and 50 U/ml, respectively.

The kinase inhibitor library, composed of 149 selective or broad-spectrum kinase inhibitors dissolved in DMSO (10 mM stock concentration), was purchased from Cayman Chemicals (#10505). The following reagents were used for cell treatments at given concentrations: LPS (Escherichiacoli O55:B5, #L2880, Sigma-Aldrich, 100 ng ml⁻¹), recombinant human TNFα (rHuTNF, #50435.50, Biomol, 10 ng/ml), Birinapant/SM (#HY-16591, MedChem Express, 1 µM), pan-caspase inhibitor zVAD-fmk (#4026865.0005, Bachem, 25 μM), MK2 inhibitor PF3644022 (#4279, Tocris, 5 μM), RIPK1 inhibitor Nec-1 (#BML-AP309-0020, Enzo Life Sciences, 50 µM), RIPK1 inhibitor Nec-1s (# 10-4544-5 mg, Tebu-Bio, 50 µM), RIPK3 inhibitor GSK872 (#HY-101872, MedChem Express, 5 µM), IKKβ inhibitor BMS345541 (#Axon 1731, Axon Medchem, 5 µM), TBK1/IKKε inhibitor BX795 (#T1830, Tebu-Bio), 5-Iodotubercidin (#HY-15424, MedChem Express, 2.5–10 µM), ABT-702 dihydrochloride (#HY-103161, MedChem Express, 5 µM), Staurosporine (#81590, Cayman, 5 µM), Etoposide (#E1383, Sigma, 5 µM), Doxorubicin (#15007, Cayman, 5 µM), Gemcitabine (#S1714, Selleckchem, 100 nM).

5AzadC, 5-FU, oxaliplatin, irinotecan, doxorubicin, and etoposide were obtained from Sigma (St. Louis, MO, USA). zVAD, Nec-1, DPQ, and PD150606 were obtained from Enzo Life Sciences (Farmingdale, NY, USA). LBW242 and BV6 are kind gifts from Novartis (Cambridge, MA, USA) and Genentech (San Francisco, CA, USA), respectively.

Cells were seeded into a 96-well plate (6 × 10⁴/well) on F-12K medium and treated with statin (10–20 μM, Merck Millipore, Germany) for 48 h, beginning 24 h after seeding. WST-1 solution (10 μL, Roche, Germany) was added to each well and the cells were incubated for 1 h. The absorbance at 440 nm was measured using a microplate reader (FLUOstar Galaxy, Germany). In some experiments, the cells were pretreated with various inhibitors such as necrostatin-1 (Nec-1, an inhibitor of RIP1, 10 μM, Enzo Life Sciences, USA), necrosulfonamide (NSA, an inhibitor of MLKL, 1.0 μM, Millipore), bafilomycin A1 (Baf-A1, 2 nM, Sigma-Aldrich), 3,4-dihydro-5[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinoline (DPQ, 60 μM, Calbiochem), Z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk, 2 μM, BioVision, USA), and mTOR inhibitors (rapamycin, 20 nM, Adooq BioScience, USA) before statin treatment.