Nature - USA (2020-01-02)

4

nature research | reporting summary

October 2018

p/557815,

Alexa Fluor® 488-conjugated antibody against NF-κB p65 phosphorylated at S529: http://www.bdbiosciences.com/eu/

applications/research/intracellular-flow/intracellular-antibodies-and-isotype-controls/anti-human-antibodies/alexa-fluor-488-

mouse-anti-nf-b-p65-ps529-k10-8951250/p/558421,

Alexa Fluor® 488-conjugated antibody against p38 phosphorylated at T180/Y182: http://www.bdbiosciences.com/eu/

applications/research/intracellular-flow/intracellular-antibodies-and-isotype-controls/anti-rat-antibodies/alexa-fluor-488-mouse-

anti-p38-mapk-pt180py182-36p38-pt180py182/p/612594,

Alexa Fluor® 647-conjugated mouse IgG1κ: http://www.bdbiosciences.com/eu/reagents/research/antibodies-buffers/

immunology-reagents/anti-human-antibodies/cell-surface-antigens/alexa-fluor-647-mouse-igg1-isotype-control-mopc-21/

p/557714,

Alexa Fluor® 488- Mouse IgG1κ: http://www.bdbiosciences.com/eu/reagents/research/antibodies-buffers/cell-biology-reagents/

isotype-controls/alexa-fluor-488-mouse-igg1-isotype-control-mopc-21/p/557782,

PE- Mouse IgG1κ: https://www.biolegend.com/en-us/products/pe-mouse-igg1--kappa-isotype-ctrl-icfc-3032,

Percp-cy5.5-Rat IgG1κ: https://www.biolegend.com/en-us/products/percp-cy5-5-rat-igg1--kappa-isotype-ctrl-4203,

V450- Mouse IgG1κ: http://www.bdbiosciences.com/eu/reagents/research/antibodies-buffers/immunology-reagents/anti-

human-antibodies/cell-surface-antigens/v450-mouse-igg1-isotype-control-mopc-21/p/560373.

Data are provided per assurance by each supplier. The commercial antibodies are well used and reported in lots of previous

publications.

The MLKL (Mus) (home made) has been validation by Wu, Z. et al. Chaperone-mediated autophagy is involved in the execution of

ferroptosis. Proceedings of the National Academy of Sciences 116, 2996, doi:10.1073/pnas.1819728116 (2019).

Eukaryotic cell lines

Policy information about cell lines

Cell line source(s) HEK293T cell line was from the American Type Culture Collection. Ripk1 gene knock-out MEFs were established from Ripk1-/-

mice. MEFs derived from D325A knockin mice were kindly contributed by Jianke Zhang.

Authentication Cell line from ATCC has been authenticated by ATCC. The primary lines are cultured for limited number of passages.

Mycoplasma contamination Cell lines tested negative for Mycoplasma contamination.

Commonly misidentified lines

(See ICLAC register)

No commonly misidentified cell lines were used.

Human research participants

Policy information about studies involving human research participants

Population characteristics The patients characteristics have been described in Extended Data Table 1. Healthy controls are less than 10 years old and they^

had no symptoms of inflammation when sampling.

Recruitment Patient samples were obtained from patients with early onset autoinflammatory disease but without clear genetic diagnosis. And

multiple age and gender matched healthy controls used in the experiment were randomly selected.

Ethics oversight Patient P1 was evaluated under protocols approved by the Institutional Review Board (IRB) by Children’s Hospital of Fudan

University (Shanghai, China). Patients P2, P3, P4 and P5 and their unaffected family members were evaluated at McMaster

Children’s Hospital (Ontario, Canada), and the Hospital for Sick Children (Toronto, Canada). Signed consent for their clinical

information to be shared and for research samples to be sent to Boston Children's Hospital (Boston, USA) was obtained. Ethics

clearance was received from the Institutional Review Board (IRB) at Boston Children's Hospital (Boston, USA) and from Western

Institutional Review Board. All relevant ethical regulations were followed. All patients and/or substitute decision markers

provided written informed consent.

Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry

Plots

Confirm that:

The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).

The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).

All plots are contour plots with outliers or pseudocolor plots.

A numerical value for number of cells or percentage (with statistics) is provided.

Methodology

Sample preparation We used EDTA-anticoagulated peripheral whole blood from patients and health donors. PBMCs from patients and healthy

donors were separated by lymphocyte separation medium (LSM) and SepMate tubes (Stemcell) according to the manufacturer's

instructions.