Article
Fisher, 78442) and 10% glycerol) for 10 min and centrifuged at 20,000g
for 10 min. Protein concentration was measured on the cleared lysates
by BCA protein assay kit (Thermo Fisher, 23225). Immunoprecipitation
and immunoblotting were conducted as described previously with
specific antibodies^23 ,^25.
In vitro RIPK1 cleavage assay
Unlabelled in vitro transcription and translation (IVTT) of 1 μg wild-
type and mutant RIPK1 constructs were performed in 50 μl reactions
using the TNT T7 Quick Coupled Transcription/Translation System
(Promega, L1170). The reaction was incubated with purified recom-
binant caspase-8 protein (R&D, 705-C8/CF) and then immunoblotted
with RIPK1 antibody.
Cytokine detection in serum
The concentrations of cytokines in serum were measured by BD Cyto-
metric Bead Array. Cytokine concentrations for IL-6, TNF and IL-10 in the
serum were determined by BD Cytometric bead arrays (BD Bioscience).
All data were analysed by FCAPArray V3 software (BD Biosciences).
Flow cytometry analysis of phosphorylation
For phos-flow staining, isolated PBMCs were treated with or without LPS
(1 μg ml−1) for 6 h at 37 °C, with 5% CO 2 and then permeabilized with Perm
Buffer III according to the manufacturer’s instructions (BD Biosciences).
Surface marker CD3, CD14 and CD19 (BD Biosciences) were used to gate
total T cells, monocytes and total B cells. The expression of p-STAT3,
p-p65 and p-p38 were analysed by flow cytometry. For phos-flow analy-
sis, the following antibodies were used: Alexa Fluor 647-conjugated
antibody against STAT3 phosphorylated at Y705 (BD Biosciences), Alexa
Fluor 488-conjugated antibody against NF-κB p65 phosphorylated at
S529 (BD Biosciences) and Alexa Fluor 488-conjugated antibody against
p38 phosphorylated at T180/Y182 (BD Biosciences). Isotype control
antibodies were used to normalize the background signals for intracel-
lular staining. All events were acquired on a FACS Canto II cytometer
(BD Biosciences) and analysed with FlowJo (Tree Star). Blue lines in
the Extended Data Fig. 2c indicate basal levels, orange lines indicate
LPS stimulation for 6 h and red lines indicate an isotype control. The
numbers mark the percentage of cells displaying phosphorylation of
STAT3 or p38 based on comparison with isotype control staining for
each cell type.
Intracellular cytokine staining
Intracellular cytokine staining for IL-6, TNF and IL-8 were measured in
PBMCs at baseline and following LPS stimulation. Cells were washed twice
with PBS, then treated with LPS (1 μg ml−1 per 1 × 10^6 PBMCs) and Golgi
plug (BD Biosciences) for 6 h at 37 °C, with 5% CO 2 and then permeabilized
with Perm/Fix for 30 min at 4 °C. Cells were stained by antibodies CD3-
Percp-cy5.5 (BD Biosciences), CD14-PE-CY7 (BD Biosciences), CD4-FITC
(BD Biosciences), CD19-APC (BD Biosciences), IL8-PE (Biolegend), IL10-
BV421 (Biolegend), IL6-Percp-cy5.5 (Biolegend) and TNF-V450 (Bioleg-
end). Isotype control antibodies were used to normalize the background
signal for intracellular staining. All events were acquired on a FACS Canto
II cytometer and analysed with FlowJo (Tree Star).
Cell viability assay
General cell survival was measured by the ATP luminescence assay
CellTiter-Glo (Promega). The percentage of viability was normalized
to readouts of untreated cells.
Cell death assay
Cell death was determined by ToxiLight Non-destructive Cytotoxicity
BioAssay Kit (Lonza, LT07) or SYTOX Green Nucleic Acid Stain (Thermo
Fisher, S7020). All experiments were conducted on 384-well plates
with at least three biological replicates. Data were collected by the
multimode plate reader (Bio Tek).
Intracellular ROS detection
Cells were seeded in 12-well plates and treated with the indicated stim-
uli for the indicated amount of time. After cell death induction, 5 μM
carboxy-H 2 DCFDA was added to cells for 30 min at room temperature.
Cells were then returned to warm growth medium and incubated for
15 min, followed by replacement of growth medium with PBS. Images
were taken using a Leica fluorescence microscope.Intracellular GSH detection
The GSH concentration in cells was assessed by GSH-Glo Glutathione
Assay Kit (Promega, V6911) according to the manufacturer’s instruc-
tions.Statistics
No statistical methods were used to predetermine sample size. For
cell-based experiments, biological triplicates were performed in each
single experiment in general, unless otherwise stated. All values were
expressed as mean ± s.e.m. and calculated from the average of at least
three independent biological replicates unless specifically stated.
Statistical analysis was performed using GraphPad Prism 8 software
(GraphPad Software). For comparisons between two groups, the Stu-
dent’s t-test (unpaired and two-tailed) was applied. In all tests, a 95%
confidence interval was used, for which P < 0.05 was considered a sig-
nificant difference. Statistical analysis of single-cell RNA sequencing
and RNA sequencing was performed using R Software (R v.3.5.2).URLs
ANNOVAR, http://annovar.openbioinformatics.org/en/latest/user-
guide/download/; CADD, https://cadd.gs.washington.edu/; gnomAD,
https://gnomad.broadinstitute.org/; Kaviar genomic variant database
(Kaviar), http://db.systemsbiology.net/kaviar/; Sorting Intolerant from
Tolerant (SIFT), https://sift.bii.a-star.edu.sg/; PolyPhen-2, http://genet-
ics.bwh.harvard.edu/pph2/; likelihood ratio test (LRT), http://www.
genetics.wustl.edu/jflab/lrt_query.html; MutationTaster, http://www.
mutationtaster.org/.Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this paper.Data availability
Gel source data are shown in Supplementary Fig. 1. Source Data for
Figs. 2–4 and Extended Data Figs. 1, 2, 4–6 are provided with the paper.
All other data that support the findings of this study are available from
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Acknowledgements We thank the patients, their families and the unaffected controls for their
support during this research study. We thank J. Zhang for Ripk1+/+Ripk3−/− and Ripk1D325A/D325A
Ripk3−/− MEFs. We thank L. Shen, M. Shi, M. Yu and R. Wang for help. Q.Z. received the grants
2018YFC1004903 from National Key Research and Development Project, 31771548 and
81971528 from The National Natural Science Foundation of China, LR19H100001 from Zhejiang
Provincial Natural Science Foundation of China and 2018QN81009 from the Fundamental
Research Funds for the Central Universities. X.W. received the grant 81373221 from The
National Natural Science Foundation of China. The work of Z.W., H.P. and X.H. was supported
by the National Key R&D Program of China (2016YFA0501900) and the China National Natural