Extended Data Fig. 6: Patient fibroblasts were resistant to both necroptosis and ferroptosis.

a, Patient fibroblasts were resistant to necroptosis induced by SM-164, Z-VAD-FMK and TNF or LPS. Fibroblasts from P1 and seven paediatric unaffected controls were treated as indicated for 24 h. LPS, 1 μg ml⁻¹; N, 10 μM Nec-1s; S, 50 nM SM-164; T, 50 ng ml⁻¹ TNF; Z, 50 μM Z-VAD-FMK. Cell death was measured by ToxiLight assay. Data are mean ± s.e.m. Circles correspond to each tested individual. Analysis of each sample was performed in triplicate. b, Patient fibroblasts showed reduced necroptosis signals after SM-164, Z-VAD-FMK and LPS stimulation compared to six paediatric unaffected controls. Patient and control fibroblasts were treated with indicated stimulation for 6 h (concentrations as in a). Cells were lysed and analysed by immunoblotting with indicated antibodies. For gel source data, see Supplementary Fig. 1. Results are representative of three independent experiments. c, Patient fibroblasts showed reduced necroptosis signals after SM-164, Z-VAD-FMK and TNF stimulation compared with a paediatric unaffected control. Patient and control fibroblasts were treated as indicated for 6 h (concentrations as in a). Cells were lysed and analysed by immunoblotting with indicated antibodies. For gel source data, see Supplementary Fig. 1. Results are representative of three independent experiments. d, The reduction of RIPK1 was rescued by Nec-1s in patient fibroblasts. Fibroblasts were treated as indicated for 24 h (concentrations as in a). NSA, 0.5 μM necrosulfonamide. Cell lysates were analysed by immunoblotting using indicated antibodies. For gel source data, see Supplementary Fig. 1. Results are representative of three independent experiments. e, Patient fibroblast showed reduced transcription levels of RIPK1 compared to five paediatric unaffected controls. The mRNA levels of RIPK1 were measured by qPCR. Data are mean ± s.e.m. Circles correspond to each tested individual. Analysis of each sample was performed in triplicate. f, Patient fibroblasts exhibited reduced TNFR1 expression at baseline compared to five paediatric unaffected controls. For gel source data, see Supplementary Fig. 1. Results are representative of three independent experiments. g, Patient fibroblasts displayed downregulation of genes involved in cell death compared with three paediatric unaffected controls. Analysis of each sample was performed in duplicate. h, Patient fibroblasts were resistant to erastin- or RSL3-induced ferroptosis compared with three paediatric unaffected controls. Cell death was measured by ToxiLight assay. Data are mean ± s.e.m. Circles correspond to each tested individual. Analysis of each sample was performed in triplicate.

Source Data