Gsk 872

Human astrocytes (HAs) were treated with 200 μM Cort for 1 h to induce cell injury. To observe the effects of necroptotic kinase inhibitors on the HA injury model, the cells were incubated with medium containing 100 μM Nec-1 (Selleck, S8037), 10 μM GSK872 (Millipore, 530389) or 1 μM NSA (Bio-Techne, 5025/10). HA was treated with 1 μM FLX during Cort treatment to estimate the protective function of FLX in the HA injury model.

Human RPE cells (ARPE-19, CLR-2302, ATCC, Manassas, VA, USA) were cultured in DME/F-12 medium (HyClone, Logan, UT, USA) supplemented with 10% FBS (HyClone) and 1× Penicillin-Streptomycin solution (HyClone) at 37 °C in 5% CO₂. Cells were treated with: 50 μM z-VAD (Sigma-Aldrich, St Louis, MO, USA), 200 μM necrostatin-1, -5, -7 (Enzo Life Sciences, Farmingdale, NY, USA), 50 μM caspase-1 inhibitor Ac-YVAD (Sigma-Aldrich), or 3 μM GSK’872 (Millipore, Billerica, MA, USA). Cells were treated with 10 mM of sodium iodate (Sigma-Aldrich) in PBS (Gibco).
Cell transfection was performed using Lipofectamine LTX (Life Technologies, Farmingdale, NY, USA). Briefly, 1 μg of HMGB1-YFP, ANT1-RFP, RIPK3-GFP, or PYCARD-GFP plasmid DNA was mixed with 5 μl of Lipofectamine LTX. The complex was added to the ARPE-19 cell cultured in 4-chamber glass slide 20 min later. Expression of the recombinant proteins was visualized upon NaIO3 treatment after 24 h under a fluorescent microscope.
To assess cell viability, MTT assay was performed as described previously.^{60 (link)} In short, ARPE-19 cells were incubated with 1 mg/ml of MTT reagent (Sigma-Aldrich) for 2 h in standard cell culture conditions. Developed MTT crystals were dissolved in DMSO (Sigma-Aldrich) and analyzed at the 96-well plate reader by measuring absorbance at 540 nm.

Neutrophils were infected in vitro with L. infantum promastigotes stationary-phase at a ratio of 1:2 (neutrophil:parasites). For assays of cell death, mouse and human neutrophils were pretreated for 30 min with zVAD-fmk (100 µM) (R&D Systems, Minneapolis, MN, USA) or zIETD-fmk (100 µM) (R&D Systems, Minneapolis, MN, USA) to block caspase activation before infection. In some experiments, Nec-1 (50 µM), GSK’872 (3 µM), or NSA (10 µM), necroptosis inhibitors (all from Merck Millipore’s Calbiochem^®, Darmstadt, Germany) were used. DMSO (vehicle) 0.4% (Cayman Chemical; Ann Arbor, MI, USA) was used as control. After 18 h, mouse infected neutrophils, or after 3 h, human infected neutrophils, were centrifuged, supernatants containing noninternalized promastigotes were collected, and medium was replaced by 250 µl Schneider insect medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 20% inactive FBS, 2 mM l-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin. After that, infected neutrophils were cultured at 25°C for an additional 3 days and intracellular load of L. infantum was estimated by production of proliferating extracellular motile promastigotes in Schneider medium (43 (link)).

HPDLFs were from 10 to 18-year-old patients who needed orthodontic extraction of healthy premolars. Patients and their parents were informed of the purpose of the experiment. Tissue explants obtained from the middle third of the root were cultured in DMEM (Gibco, USA) with 20% fetal bovine serum (Gibco, Australia) and 10% penicillin/streptomycin solution (Life Technologies). Cells at the third to sixth passage were used in the experiment. Necrostatin-1(Nec-1) (Selleckchem, USA), GSK’872 (Merck-Millipore, Germany) and necrosulfonamide (NSA) (Enzo Life Sciences, USA) were utilized to block RIPK1, RIPK3 and MLKL, respectively. The PDLFs were pretreated with these inhibitors for 2 hours and then stimulated with P. gingivalis.
THP-1 human monocytic cells (ATCC) were grown in RPMI 1640 medium (Gibco) with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin solution. Cells were treated with P. gingivalis of different multiplicity of infection (MOI) and continued to be incubated at 37 °C, 5% CO2 under the normal oxygen condition in a humidified environment.

The Smac mimetic AZD5582 was obtained from Chemietek (Indianapolis, IN, USA) and Smac mimetics SM164, BV6, and Birinapant (TL32711) were from APExBIO (Houston, TX, USA). Recombinant human IFNα was from PBL Assay Science (Piscataway, NJ, USA) and IFNγ, IFNλ, TNFα, and Annexin V-FITC were from eBioscience (San Diego, CA, USA). Recombinant human TRAIL was from ProSpec TechnoGene (East Brunswick NJ, USA). Polyinosinic–polycytidylic acid (poly(I:C)) was from InvivoGen (San Diego, CA, USA). Necrostatin-1, necrosulfonamide, GSK872, Bay11-7082, JAK kinase inhibitor I, AG-1478, and cisplatin were from EMD Millipore (Billerica, MA, USA). The general caspase peptide inhibitor Z-VAD-FMK and the caspase-8 peptide inhibitor Z-IETD-FMK were from R & D Systems (Minneapolis, MN, USA). Human TNFα neutralizing antibody (#7321) was from Cell Signaling Technology (Beverly, MA, USA). Caspase-3 and -8 colorimetric assay kits were from BioVision (Milpitas, CA, USA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).