Etanercept enbrel

Etanercept (Enbrel®), a recombinant human p75 TNF receptor fused to an IgG constant fragment, was purchased from Pfizer (New York, NY, USA) and diluted with saline (Otsuka Pharmaceutical Factory, Tokushima, Japan) for subcutaneous administration (0.4 mg/mL). Tacrolimus was purchased from MedChemExpress (Monmouth Junction, NJ, USA) and suspended in 1% methylcellulose (Tokyo Chemical Industry, Tokyo, Japan) solution for oral administration (0.3 mg/mL).

PMKs were obtained from Hoip^E-KO newborn pups, Tnfr1^KO;Hoip^E-KO and Tnfr1^KO;Hoil-1^E-KO adult tails according to established protocols^{64 (link)}. Briefly, skin was incubated with 0.25% Trypsin in Hank’s Balanced Salt Solution without calcium and magnesium (Stratech Scientific Ltd) overnight at 4 °C. On the following day, the dermis and epidermis were separated. Cell suspensions were cultured in Eagle’s minimal essential medium (Lonza) without calcium with 8% chelate foetal calf serum and penicillin–streptomycin (Sigma). PMKs were seeded in plates pre-coated with collagen I (Life technologies) for subsequent experiments. PMKs were cultured in medium supplemented with 20 µM Z-VAD-fmk (Abcam), 10 µM Necrostatin-1s (Cambridge Bioscience), 1 µM RIPK3 inhibitor (GSK2399872B) or 50 µg mL⁻¹ Etanercept (Enbrel®) (Pfizer and Pentaglobin from Biotest) for 4 days, with supplemented medium replaced every day. On the last day, cell viability was measured using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega) following the manufacturer’s instructions. Alternatively, PMKs were treated for 24 h with the following ligands as indicated: 50 ng mL⁻¹ mouse iz-TRAIL, 50 ng mL⁻¹ CD95L-Fc, or 100 µg mL⁻¹ Poly(I:C) (Invitrogen).