Fig. 5: Phenytoin prevents the formation of the necrosome.

a HT29 cells were treated with TSZ for 4 h and stained for human pMLKL. Note that phenytoin prevents the phosphorylation of MLKL as effectively as Nec-1s. b Screen for kinase inhibition by phenytoin reveals no major inhibition of any of the kinases investigated, including RIPK1 and RIPK3. c RIPK1 autophosphorylation assay as described in detail in the Methods section. 250 µm phenytoin result in 43% inhibition of RIPK1 autophosphorylation; Nec-1s serves as positive control. d HT-29 cells were treated with TSZ and phenytoin as indicated and NP40 soluble and insoluble fractions were separated before RIPK1 western blotting. e RAW cells were treated with LPS/zVAD-fmk (L/Z), phenytoin, Nec-1s and the RIPK3-inhibitor GSK872 as indicated. NP40 soluble and insoluble fractions were separated and stained for RIPK1 (short and long exposure), RIPK3 and pMLKL. f, g Primary wild-type MEFs were treated with TNF-α (50 ng/ml) in the presence of CHX (200 ng/ml) and zVAD (50 μm), with or without pre-treatment with indicated doses of phenytoin or RIPK1 inhibitor GSK’963 (indicated doses in (f), 5 µm in g) or Nec-1 (50 μm) or RIPK3 inhibitor GSK’872 (5 μm). pMLKL western blotting and RIPK3 immunoprecipitations were performed on lysates after 6 h and examined for necrosome formation by immunoblotting for RIPK1. h L929 cells were seeded on 100 mm plate and then stimulated with hTNF (10 ng/ml) in the presence/absence of zVAD-fmk (10 μm) and phenytoin (500 μm) for 2 and 4 h. Cells were then lysed in NP-40 lysis buffer (0.2% NP-40, 20 mm Tris, 150 mm NaCl and 10% glycerol, at pH 7.5) for 30 min in ice. Samples were centrifuged at 20,000 × g for 15 min and supernatants were incubated with FADD-specific antibody (M-19, Santa Cruz Biotechnology) overnight at 4 °C. Protein A/G beads (Santa Cruz Biotechnology) were added for a further 3 h. The beads were then washed five times with cold lysis buffer and FADD-associated proteins were eluted following incubating the beads in SDS-PAGE loading buffer at 70 °C for 20 min. i, j NIH3T3 + RIPK3-2xFV were incubated in absence or presence of 0.125 mm phenytoin in combination with 10 ng/ml of TNF plus 25 µm zVAD (g) or 2 nm AP-1 (homodimerizer; AP-20187) (h). Cell death was monitored by Sytox Green uptake by using an incucyte Kinetic Live Cell Imager. Alternatively, cell death was prevented by using 30 µm of Nec-1s (RIPK1 inhibitor) or 1 µm GSK’872 (RIPK3 inhibitor), respectively. k KBGFP#2 cells were seeded on six-well plate and then stimulated with hTNF (10 ng/ml) in the presence/absence of phenytoin (either 250 μm or 500 μm) for 24 h. Cells were then trypsinized and washed once with cold PBS. The GFP expression was analyzed by flow cytometry