Extended Data Figure 4: In vivo and in vitro characterization of cell death in human LUBAC deficiency.

(a) Hematoxylin and eosin (H&E) staining of colon biopsy samples from LUBAC-deficient patients. Bars: 0.4mm. These images were obtained as a part of clinical testing and were not repeated. (b-c) Supporting data for Fig. 3g to validate the specificity of p-RIPK1 antibody to detect RIPK1 Ser166 phosphorylation with (b) western blot and (c) immunocytochemistry. HT29 cells were stimulated with TSZ (TNF + smac mimetic (BV6) + zVAD) for 4h. Note that TSZ-stimulated cells show positive staining of pRIPK1, which was removed by λ-phosphatase treatment. (d-e) Supporting data for Fig. 3h to validate the antibody specificity for cleaved GSDMD (Asp275) with (d) western blot and (e) immunocytochemistry. THP1 cells were pre-incubated with LPS for 3h and were further stimulated with nigericin for another 1h. (b-e) These experiments were aimed to confirm the specificity of the antibodies and were not repeated. (f) Cleavage of GSDME in dermal fibroblasts stimulated with TNF (100 ng/ml) + CHX (50 μg/ml) for the indicated times. HeLa cells were used as a positive control. Representative result of two independent experiments.