www.sciencemag.org/content/science.1249361/DC1

Supplementary Material for

Activity of Protein Kinase RIPK3 Determines whether Cells Die by Necroptosis or Apoptosis

Kim Newton,* Debra L. Dugger, Katherine E. Wickliffe, Neeraj Kapoor, M. Cristina de-Almagro, Domagoj Vucic, Laszlo Komuves, Ronald E. Ferrando, Dorothy M. French,

Joshua Webster, Merone Roose-Girma, Søren Warming, Vishva M. Dixit*

*Corresponding author. E-mail: knewton@gene.com (K.N.); dixit@gene.com (V.M.D.)

Published 20 February2014 on Science Express

DOI: 10.1126/science.1249361

This PDF file includes:

Materials and Methods

Figs. S1 to S6

Full Reference List

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Materials and Methods Mice Constructs for gene targeting of ES cells were made using recombineering and/or other established molecular biology methods. Taconic (Germany) generated Ripk3LSL/+ mice using C57BL/6NTac ES cells. genOway (France) generated Ripk1D138N/+ mice using C57BL/6 ES cells. Ripk3D161N/+, Ripk1+/-, and Casp8+/- mice were generated at Genentech using C57BL/6N C2 ES cells. All alleles were maintained on a C57BL/6N genetic background. The Ripk1 deleted region corresponds to genomic position (NCBI37/mm9 assembly): chr13:34,101,233-921. The Casp8 deleted region corresponds to: chr1:58,883,729-58,884,429. The Ripk3LSL allele has a LSL cassette inserted in intron 3 at position chr14:56,406,479 (reverse strand). Ripk3-/- mice were described previously (6) although the mice used in this study had been deleted of the loxp-flanked neomycin selection cassette through breeding to the general cre deleter strain C57BL/6-Gt(ROSA)26Sortm16(cre)Arte (Taconic). Dai-/- (19), Cyld-/- (20), Tnfr1-/- (21), Trif-/- (22), Dr3-/- (23) and Mlkl-/- (24) mice have been described previously. Flip conditional knock-out mice (25) were bred to the cre deleter strain C57BL/6-Gt(ROSA)26Sortm16(cre)Arte to obtain Flip+/- mice. Ripk1 genotyping primers: 5’ CAG GGG CAG CAG TGC TAT GG and 5’ CAT GTC AGC CTT GCA AGC AGT G amplified 408 bp WT and 578 bp Ripk1D138N DNA fragments; 5’ GAT GCC AGA CAT GAA GCA TCC, 5’ GGA GGA TTA AAG GTC TGA GGC, and 5’ TCA TTG CCT CCC TTA CTA CCC amplified 265 bp WT and 231 bp Ripk1- DNA fragments. Casp8 genotyping primers: 5’ CAG TGG ATG CAT GCA AG G, 5’ TCT CCT GAA GAT GGG TCA CC, and 5’ GGA GCC AGT ATT CTG CTA GCA A amplified 349 bp WT and 304 bp Casp8- DNA fragments. Ripk3 genotyping primers: 5’ AGA AGA TGC AGC AGC CTC AGC T, 5’ ACG GAC CCA GGC TGA CTT ATC TC, and 5’ GGC ACG TGC ACA GGA AAT AGC amplified 130 bp WT and 298 bp Ripk3- DNA fragments; 5’ CGC TTT AGA AGC CTT CAG GTT GAC and 5’ ACG GAC CCA GGC TGA CTT ATC TC amplified 345 bp WT and 379 bp Ripk3D161N DNA fragments; 5’ ACG CCA AGG TTA GTC CAT CTA, 5' ACC AGA GGG AAG GTA AAG TCA and 5’ CGG TTA CCA TAA CTT CGT ATA GCA amplified 278 bp WT, 235 bp Ripk3LSL and 324 bp Ripk3ΔLSL DNA fragments. Ripk3LSL/+ mice were crossed to the inducible general Cre deleter C57BL/6-Gt(ROSA)26Sortm9(Cre/ESR1)Arte (Taconic). The CreERT2 allele was maintained in a heterozygous state. CreERT2 was activated in mice by intraperitoneal injection of 40 mg/kg tamoxifen dissolved in sunflower seed oil daily for 5 consecutive days. WT, Ripk3-/-, and Ripk1D138N/D138N mice were dosed with 300 µg/kg mouse TNF (R&D Systems) intravenously via the tail vein. Body temperature was determined every 2 h for the first 12 h via a lubricated digital rectal probe. Mice with a body temperature below 23.6°C or that were moribund were euthanized. For timed pregnancies, embryos were designated E0.5 on the morning a vaginal plug was detected. The Genentech animal care and use committee approved all mouse protocols.

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Immunofluorescence and confocal microscopy Yolk sacs were fixed in PBS containing 4% paraformaldehyde for 2 h at 4°C, washed in PBS, permeabilized in PBS containing 0.25% Triton X-100 for 45 min, and blocked in PBS containing 0.1% Triton X-100 and 2% donkey serum for 1 h. Sacs were stained in the same blocking buffer with rat anti-CD31 (BD Biosciences cat #550274 diluted 1:50) and rabbit anti-cleaved caspase-3 (Cell Signaling Technology cat #9661 diluted 1:500) antibodies overnight at 4°C. After 3 washes in PBS containing 0.1% Triton X-100, yolk sacs were stained with Cy3-labeled donkey anti-rabbit IgG (Jackson ImmunoResearch cat# 711-165-152 diluted 1:300 in blocking buffer) for 2 h at room temperature. After a further 3 washes, yolk sacs were stained with Cy5-labeled donkey anti-rat IgG (Jackson ImmunoResearch cat# 712-175-153 diluted 1:300 in blocking buffer) for 2 h at room temperature. The tissues were washed in PBS 3 times and mounted with Prolong Gold anti-fade reagent and DAPI (Life Technologies). A LEICA LSI laser-scanning confocal microscope was used to acquire single optical sections, using a 5x objective with a 1.5x optical zoom, resulting in a 128x final magnification, imaging a 1.18 mm x 1.18 area. All images were acquired with a 1.56 Airy unit (199.3 micrometer) pinhole size resulting in 2.3 micron optical section thickness. For the detection of the Cy3 channel, a 532 laser (48% output) was used with the following detector settings: 542-598 nm detector range, 979 gain and -16 offset values. For localization of the Cy5 signal, a 635 laser was used (81% power output), 969 gain, -20 offset values, and 653-719 nm detector range. The LUT for the Cy5 channel was set to green. Image analysis was performed using a custom journal written in Metaxpress 3.1 software (Molecular Devices). Merged color images were separated into their grayscale channel components. The number of cleaved caspase-3 positive cells was counted using the nuclei detection application. Measurements were normalized to the field of view. Cell cultures MEFs, BMDMs and T-cells were cultured in the high glucose version of Dulbecco’s Modified Eagle Medium supplemented with 10% heat inactivated-fetal calf serum (HI-FCS), 2 mM glutamine, 100 µM asparagine, 55 µM 2-mercaptoethanol, 50 U/mL penicillin, and 50 µg/mL streptomycin. Primary MEFs derived from E13.5 or E14.5 embryos were immortalized with the retroviral vector pWZL-hygro-E1A. BMDMs were differentiated from bone marrow cells in non-treated plates and medium supplemented with 25 ng/mL mouse M-CSF (R&D Systems) for 5 days. Adherent BMDMs then were stimulated in the continued presence of M-CSF with 100 ng/mL mouse TNF (R&D Systems) and/or 20 µM zVAD.fmk (Promega). Where indicated, MEFs were stimulated with 100 ng/mL mouse TNF, 20 µM zVAD.fmk, 30 µM necrostatin-1 (Sigma), or 100 nM 4-hydroxytamoxifen (Sigma). T-cells were purified by negative selection in a FACSAria (BD Biosciences) after staining lymph node cells with FITC-conjugated antibodies to B220, CD19, IgM, IgD, Gr-1, Mac-1, and TER-119 (BD Biosciences). Cells were stained with propidium iodide (PI; BD Biosciences) to exclude dead cells. T-cell purity was 94-98% when sorted cells were stained with antibodies to CD4 and CD8. T-cells were cultured at 2x105 cells/mL with plate bound 145-2C11 anti-CD3 and 37.51 anti-CD28 antibodies (BD Biosciences; 10 µg/mL of each antibody in the

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coating solution). 100 µL cultures were pulsed with 0.5 µCi [methyl-3H]-thymidine (PerkinElmer) for 6 h on the days indicated. Survival assays and flow cytometry Adherent BMDMs were harvested with a cell scraper into the culture medium that also contained any floating dead cells. Adherent MEFs were harvested by trypsinization and pooled with dead floating cells in the culture supernatant. Cell suspensions were stained with 2.5 µg/mL PI and analyzed in a FACSCanto II (BD Biosciences) for the % of PI-negative viable cells.

Single cell suspensions were prepared from lymph nodes (mesenteric, inguinal, axillary, and brachial) with 40 µm cell strainers (BD Biosciences). Cells were stained with APC-conjugated anti-B220 (BD Biosciences, clone RA3-6B2) and FITC-conjugated anti-CD3 (BD Biosciences clone, 145-2C11) antibodies in PBS containing 2% HI-FCS and 1% rat serum. After washing, cells were resuspended in PBS, 2% HI-FCS and 2.5 µg/mL PI for analysis in a FACSCanto II.

Immunoprecipitation and western blotting Cells were lysed in 20 mM Tris.HCl pH 7.5, 135 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton X-100, 10% glycerol, 1 mM DTT, phosSTOP phosphatase inhibitors (Roche) and a complete protease inhibitor cocktail (Roche). FADD was immunoprecipitated from the soluble lysate overnight at 4°C with rabbit anti-FADD antibody (Genentech, clone 1.28E12) and protein A/G-agarose beads (Pierce). Beads were washed extensively in 50 mM Tris.HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and protease/phosphatase inhibitors and then eluted by heating in LDS sample buffer (Life Technologies). Antibodies used for western blotting recognized RIPK1 (BD Biosciences), RIPK3 (Genentech rat clone 1G6.1.4), Caspase-8 (Enzo Life Sciences, rat clone 3B10), MLKL (rat monoclonal a gift of W. Alexander, Walter and Eliza Hall Institute, Australia), Flag (M2, Sigma), β-actin (MP Biomedicals, mouse clone C4) phosphorylated IκBα, IκBα, phosphorylated JNK, JNK, phosphorylated ERK, and ERK (Cell Signaling Technology, cat #9241, 9242, 9101, 9102, 9251, and 9252). In vitro kinase assays

293T cells transfected with 3xFlag-tagged mouse RIPK1 or RIPK3 plasmid constructs were lysed in TBS supplemented with 0.1% Triton X-100 and protease inhibitor tablets (Roche). RIPK proteins were immunoprecipitated from the soluble lysate with M2 anti-Flag agarose beads (Sigma) by rotation at room temperature for 15 min. The beads were washed in TBS three times and resuspended in 30 µL of reaction buffer (50 mM Tris.HCl pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM MnCl2, and 2 mM DTT). 5 µCi [γ-32P]-ATP (Perkin Elmer) was added in a volume of 5 µL and reactions were incubated at 30°C for 30 min. Proteins were eluted from the beads by heating in LDS sample buffer (Life Technologies). Immunohistochemistry

Formalin-fixed, paraffin-embedded 4 µm tissue sections were deparaffinized in xylenes and rehydrated through a graded series of alcohols. Antigen retrieval was in Target Retrieval Solution (DAKO) for 20 min at 99°C in a PT Module (Thermo

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Scientific). After a 20 min cool down, endogenous peroxidase activity was quenched with KPL blocking solution (Kirkegaard and Perry Laboratories) for 4 min. Endogenous avidin/biotin was blocked with an avidin/biotin blocking kit (Vector Labs). Non-specific binding sites were blocked for 30 min with PBS containing 10% rabbit serum and 3% BSA (RIPK3 staining) or PBS containing 10% donkey serum and 3% BSA (RIPK1 staining). Sections were stained for RIPK1 with 0.5 µg/mL rat 10C7.3.1 anti-RIPK1 antibody (Genentech) for 1 h at room temperature, followed by a biotinylated donkey anti-rat secondary antibody (Jackson ImmunoResearch) for 30 min, Vectastain Elite ABC-HRP reagent (Vector Labs) for 30 min, and then metal enhanced DAB peroxidase substrate (Pierce) for 5 min. Sections were stained for RIPK3 with 5 µg/mL rat 1G6.1.4 anti-RIPK3 antibody (Genentech) and a biotinylated rabbit anti-rat secondary antibody (Vector Labs). Sections were counterstained with Mayer’s hematoxylin (Rowley Biochemical Inc.). Taqman assays RNA was isolated from MEFs with a RNeasy mini-kit (Qiagen). An on-column DNase treatment was included. RNA content was evaluated in a Nanodrop ND1000. RNA quality was assessed in an Agilent 2100 Bioanalyzer using the Agilent RNA 6000 Pico total RNA kit. cDNA was generated from each RNA sample using a Taqman Gene Expression Cells to Ct kit (Life Technologies). Gene expression assays were from Life Technologies: Ripk3 Mm01319233_g1; b-actin Mm00607939_s1. Experiments were performed in triplicate using the standard curve method. Ripk3 levels were normalized against β-actin gene expression.

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A 3xFlag.RIPK3

WT

D1

61

N

150250

100

75

50

37

25

20

15

150250

100

75

50

37

25

20

15

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BWT Ripk3 allele

1 2 3 4

ATG

Ripk3D161N allele

1 2 3 4

GAT (asp) > AAT (asn)

frt

loxp

C Ripk3+/+

Ripk3

+/+

Ripk3

-/-

Ripk3LSL allele

1 2 3 4

GAT (asp) > AAT (asn)

STOP

IP Flag

WB Flag

IP Flag32P autoradiograph

Fig. S1. Generation of a Ripk3D161N knock-in allele. A. Western blot of RIPK3 immunoprecipitated from transfected 293T cells (upper panel). Autoradiograph after immunoprecipitated RIPK3 was used in an in vitro kinase assay (lower panel). WT, wild-type. B. Organization of Ripk3 gene-targeted alleles. C. E9.5 embryos. Cells expressing RIPK3 are stained brown. Left panel: scale bar, 200 µm. Right panels: scale bar, 500 µm.

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AWT Casp8 allele

2 3

ATG

Casp8- allele

4

2

ATG

4loxp

C

1.6%

36%

B220

60%1 71%

16%

WT Ripk3D161N/D161N Casp8-/-

BRipk3+/D161N

Casp8+/-Ripk3D161N/D161N

Casp8+/-Ripk3D161N/D161N

Casp8-/-

(n=5)

4

-/- D

0 1 2 3

WT (n=4)

Ripk3D161N/D161N Casp8

103

104

105

106

[3H

]-th

ym

idin

e

Upta

ke (

cpm

)

Day

Aged 6 weeks

66% 0.6%

31%

55% 7%7%

35%

11%

CD

3

Aged 8 weeks

Aged 18 weeks Aged 19 weeks

Fig. S2. Effect of caspase-8 deficiency on Ripk3D161N/D161N mice. A. Organization of the Casp8 knock-out allele. WT, wild-type. B. E15.5 littermates. C. Dot plots of lymph node cells stained for CD3 and B220 and then analyzed by flow cytometry. D. [3H]-thymidine uptake by purified T-cells cultured with plate-bound antibodies to CD3 and CD28. Data represent the mean ± sd.

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D

Sple

en

WT

Ripk3D161N/D161NRipk1+/-

Liv

er

C WT Ripk1-/-

SI

SI

UBUB

LV

BF

LVWT Ripk1 allele

2 3

ATG

Ripk1- alleleloxp 3

A

B

Colo

nT

hym

us

Bro

wn

Fat

WT Ripk1-/-

P0

LI LI

CRCR

TH TH

BF BF

Ripk3D161N/D161NRipk1-/-WT

E18.5

LI LI

CR

CR

TH TH

BF BF

Fig. S3. Effect of RIPK1 deficiency on Ripk3D161N/D161N mice. A. Organization of the Ripk1 knock-out allele. WT, wild-type. B. Hematoxylin and eosin stained tissues. Thymus (TH) and P0 brown fat (BF): magnification, 5x. E18.5 brown fat and P0 colon: magnification, 10x. E18.5 colon: magnification, 20x. LI, large intestine. CR, crypt. C. E18.5 embryos sections. Cells expressing RIPK1 are stained brown. Magnification, 5x. LV, liver. SI, small intestine. UB, urinary bladder. D. Hematoxylin and eosin stained tissues from E18.5 embryos.

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Dai+/+

Dai+/-

Dai-/-

Ripk3+/+

11228

Ripk3D161N/D161N

000

Ripk3D161N/+

253118

B Ripk3+/D161N Dai-/- Ripk3D161N/D161N Dai-/-

C

Trif+/+

Trif+/-

Trif-/-

Ripk3+/+

102717

Ripk3D161N/D161N

000

Ripk3D161N/+

315823

D Ripk3+/+ Trif-/- Ripk3D161N/D161N Trif-/-

A

E

Cyld+/+

Cyld+/-

Cyld-/-

Ripk3+/+

384820

Ripk3D161N/D161N

000

Ripk3D161N/+

6112643

F Ripk3+/+ Cyld-/- Ripk3D161N/D161N Cyld-/-

G

Tnfr1+/+

Tnfr1+/-

Tnfr1-/-

Ripk3+/+

987

Ripk3D161N/D161N

000

Ripk3D161N/+

152310

H Ripk3+/+ Tnfr1-/- Ripk3D161N/D161N Tnfr1-/-

I

J

K

Mlkl+/+

Mlkl+/-

Mlkl-/-

Ripk3+/+

121715

Ripk3D161N/D161N

000

Ripk3D161N/+

93115

Ripk3+/D161N Mlkl-/- Ripk3D161N/D161N Mlkl-/-

Flip+/+

Flip+/-

Flip-/-

Ripk3+/+

15240

Ripk3D161N/D161N

000

Ripk3D161N/+

32740

L

Ripk1D138N/D138N

Ripk3+/+

23

Ripk3D161N/D161N

0

Ripk3D161N/+

36

M

Dr3-/- Tnfr1-/-

Ripk3+/+

17

Ripk3D161N/D161N

0

Ripk3D161N/+

38

Ripk3+/+

Tnfr1-/- Dr3-/- Ripk3D161N/D161N

Tnfr1-/- Dr3-/-

N

Ripk3+/+

Ripk1D138N/D138NRipk3D161N/D161N

Ripk1D138N/D138NO

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Fig. S4. DAI, TRIF, CYLD, TNFR1, MLKL, FLIP, DR3, and the kinase activity of RIPK1 are dispensable for the lethality of Ripk3D161N/D161N mice. A, C, E, G, K, and M. Numbers of surviving offspring from compound heterozygote parents. B. E12.5 littermates D, F, and L. E10.5 littermates. H, J, and O. E11.5 littermates. I. Numbers of surviving offspring from Ripk3D161N/+ Tnfr1-/- Dr3-/- parents N. Numbers of surviving offspring from Ripk3D161N/+ Ripk1D138N/D138N parents.

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A

C100

80

60

40

20

0

% S

urviv

al(tr

eate

d/un

treat

ed)

4-OHTzVADNec1

+ + ++

+

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80

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0

% S

urviv

al(tr

eate

d/un

treat

ed)

TNFzVADNec1

++

+++

Ripk3LSL/LSL CreERT2 WTD

-- -

--

Days of 4-OHT

3

0

1

2

Rip

k3 m

RNA

expr

essio

n(re

lativ

e to

Rip

k3+/

+ on

day

0)

Ripk3+/+ CreERT2Ripk3LSL/LSL CreERT2

E

0 1 2 3

B Ripk3+/+ CreERT2 Ripk3LSL/LSL CreERT2

Ripk3+/+ CreERT2

Ripk3LSL/LSL CreERT2Ripk3LSL/+ CreERT2

x

0 1 2 3 4 5 616

20

24

28

Day

Body

wei

ght (

g)

Fig. S5. Effect of tamoxifen-induced RIPK3 D161N expression. A. Body weights of Ripk3+/+, Ripk3LSL/+, and Ripk3LSL/LSL mice expressing CreERT2 treated with tamoxifen on days 1-5. The x indicates that this mouse was found dead on day 6. B. Hematoxylin and eosin staining of thymus harvested 2 days after the final tamoxifen injection. Magnification, 5x. C. Survival of E1A-immortalized Ripk3LSL/LSL CreERT2+ MEFs after 2 days in culture with the reagents indicated. D. Survival of E1A-immortalized wild-type (WT) MEFs after 24 h in culture with the reagents indicated. E. Ripk3 mRNA expression in E1A-immortalized MEFs. Data represent the mean ± sd of 3 assays.

12

150250

10075

50

37

25201510

A 3xFlag.RIPK1

WT

D13

8N

BWT Ripk1 allele

2 3 4

ATG

Ripk1D138N alleleGAC (asp) > AAC (asn)

IP FlagWB Flag

IP Flag32P autoradiography

150250

100755037

25201510

2 3 4 loxplox511

C

120

80

40% S

urvi

val

(trea

ted/

untre

ated

)

TNFzVAD +

+ ++

--

WTRipk1D138N/D138NRipk3-/-

0

D

Ripk1D138N/D138N

WT

MLKL

50

50

RIPK3

-actin37

Fig. S6. Generation of Ripk1D138N/D138N mice. A. Western blot of RIPK1 immunoprecipitated from transfected 293T cells (upper panel). Autoradiograph after immunoprecipitated RIPK1 was used in an in vitro kinase assay (lower panel). B. Organization of the Ripk1D138N allele. C. Survival of E1A-immortalized MEFs after 24 h. % Survival = (% PI-negative treated cells)/(% PI-negative untreated cells) x 100. Each bar represents a different cell line. Data are the mean ± sem of 3 independent experiments. D. Western blots of primary BMDMs.

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