SUPPLEMENTARY INFORMATION

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Kelley Gibson
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infection: fla Legionell infection: B6 Nip5 -/- B6 Nip5 -/- 1 1 8 8 %LDH Relese 6 %LDH Relese 6 Supplementry igure 1.

() Immortlized C57BL/6 mcrophges were trnsduced with NAI2- knockdown lentivirus or negtive controls, cell lystes were prepred, nd equl totl

() HEK293T cells were co-trnsfected with NAI expression vectors nd.1 control vector, the NAI2 shrna constructs (NAI2 shrna#1-#3), or the.

1 SULEMENTARY INORMATION doi:1.138/nture1394 Trnsduced Mcrophges 293T-Nip trnsfectnts NAI2 N2 N5 5 Blot: Nip2 Blot: Nip5 N1 lg N6 5 c Blot: lg Blot: Nip6 Wild-type Legionell infection: fla Legionell infection: B6 Nip5 -/- B6 Nip5 -/ %LDH Relese 6 %LDH Relese 6 Supplementry igure 1. NAI2 knockdown specificlly ffects NAI2 expression nd does not ffect expression of other NAI proteins. () Immortlized C57BL/6 mcrophges were trnsduced with NAI2- knockdown lentivirus or negtive controls, cell lystes were prepred, nd equl totl protein from ech smple were seprted y SDS-AGE nd western lotted for NAI2 expression. () HEK293T cells were co-trnsfected with NAI expression vectors nd.1 control vector, the NAI2 shrna constructs (NAI2 shrna#1-#3), or the.1- negtive control for 48h, followed y western lotting for expression of NAI proteins. No nti-nai1 ntiody exists, so lg-tgged NAI1 construct ws employed. (c) NAI2 knockdown cells were infected with wild-type or flgellin-deficient Legionell pneumophil (MOI=2) nd LDH relese ws mesured 4 hours fter initil infection. Dt shown ± s.d. re representtive of t lest three independent experiments. 1

RESEARCH SULEMENTARY INORMATION 47 1 Ln-lA + A.5 1 1.5 2 3 Time (hrs.) Whole Cell Lyste WB : Cspse1 37 5 37 Ln-lA + A.5 1 1.5 2 3 4 5 6 Time (hrs.

WB : Cspse-1 () Trnsfection of 293T cells with, NAI5, nd CAS1 ws followed 18h lter y delivery of purified Ln-lA [5 ( tive of t lest three independent experiments.

2 RESEARCH SULEMENTARY INORMATION 47 1 Ln-lA + A Time (hrs.) Whole Cell Lyste WB : Cspse Ln-lA + A Time (hrs.) Whole Cell Lyste WB : IL-1 WB : β-ctin 1 Sup TCA ppt. WB : Cspse-1 () Trnsfection of 293T cells with, NAI5, nd CAS1 ws followed 18h lter y delivery of purified Ln-lA [5 ( tive of t lest three independent experiments. Trnsfected lsmids 6x-Myc-lA NAI5 I IgG Myc NAI5 11 WB 6 Myc Supplementry igure 3., NAI5, nd flgellin (la) cn e co-immunoprecipitted. 293T cells were trnsfected with NAI5,, nd 6x-Myc- la for 48 hours, nd digitonin cell extrcts were immunoprecipitted with either control IgG, nti-, or nti-myc ntiodies. Immunoprecipittes were seprted y SDS- AGE, nd Western lotted for NAI5,, or Myc. Dt shown re representtive of t lest three independent experiments. 2

SULEMENTARY INORMATION RESEARCH 4-12% 2D SDS-AGE Trnsfection: NAI5, 3-12% 1D Ntive AGE 148 48 242 146 1 WB : NAI5 Trnsfected lsmids NAI5-lg NAI5-lg G-lA (Ntive) SDS-AGE (4-12%) NAI5-lg G-lA (Dentured

loop - - + NAI5 347 + + + + + + G-lA * Oligomerized NS e Ntive AGE 1 7 + + + CARD - + + 6x-Myc-lA - - + NAI5 * Oligomerized NS Monomer Monomer WB : NAI5 11 SDS- AGE 11 SDS- AGE 5 WB : Myc 65 WB : G

3 SULEMENTARY INORMATION RESEARCH 4-12% 2D SDS-AGE Trnsfection: NAI5, 3-12% 1D Ntive AGE WB : NAI5 Trnsfected lsmids NAI5-lg NAI5-lg G-lA (Ntive) SDS-AGE (4-12%) NAI5-lg G-lA (Dentured prior to Ntive dimension) % 1D Ntive AGE WB : lg c Trnsfected lsmids 6x-Myc-lA 4-12% 2D SDS-AGE 3-12% 1D Ntive AGE WB : Myc d Ntive AGE NAI NAI5 loop NAI G-lA * Oligomerized NS e Ntive AGE CARD x-Myc-lA NAI5 * Oligomerized NS Monomer Monomer WB : NAI5 11 SDS- AGE 11 SDS- AGE 5 WB : Myc 65 WB : G WB : NAI5 WB : β-ctin WB : β-ctin Supplementry igure 4. ormtion of the inflmmsome depends on flgellin, the nd of NAI5, nd is independent of the CARD of. () NAI5 nd were expressed in 293T cells for 48 hours (without flgellin), followed y digitonin ntive cell extrct preprtion, nd seprtion y BN-AGE followed y second dimension of SDS-AGE. () NAI5-lg ws expressed in 293T cells in the sence of nd with or without G-flgellin, nd smples were treted s in, ut with western lotting to detect NAI5-lg. (c) As in, ut with expression of 6x-Myc-lA in plce of nd NAI5. (d) Wild-type NAI5, - la nd nlyzed s in igure 3. The -loop is essentil for the function of the nucleotide inding domin () e 6x-Myc-lA, or with oth 6x-Myc-lA nd NAI5 for 48 hours, followed y smple preprtion nd nlysis s in igure 3. *NS denotes non-specific nd. Dt shown re representtive of t lest three independent experi- 3

4 RESEARCH SULEMENTARY INORMATION 5 *** *** ns ns ns %G high cells wild-type (K1R) (K1R) LRR LRR CARD CAS1 NAI5 la *** ns ns ns %G high cells 3 1 NAI5 wild-type NAI5 -loop NAI5 LRR NAI5 347 CAS1 la Supplementry igure 5. Roles of nd NAI5 Domins in Responsiveness to Cytosolic lgellin. () Wild-type or mutnt constructs were trnsfected into 293T cells lone or with different comintions of CAS1, NAI5, nd la MSCV2.2-IRES-G expression vectors. orty-eight hours fter trnsfection, cells were collected, nd nlyzed y flow cytometry for G expression. The ility of (K1) to signl is surprising. Trnsduction of the (K1R) llele into Nlrc4-/- mcrophges confirmed tht the llele is indeed functionl, even in mcrophges (Supplementry ig. 6). We speculte tht lthough (K1R) cnnot uto-oligomerize it my still e le to e recruited to oligomerized NAI5 complexes nd my still e le to function s n dptor to recruit nd ctivte CAS1. () Wild-type or mutnt NAI5 constructs were trnsfected lone or in comintion with CAS1,, nd la for 48 hours, followed y nlysis for G expression. ns, not significnt;; ***, <.1. Dt shown (± s.d.) re representtive of t lest three independent experiments. 4

SULEMENTARY INORMATION RESEARCH %LDH Relese 1 8 6 *** *** *** *** - A + A / Ln-lA %LDH Relese *** *** 1 *** *** - L2 fla + L2 (la+) 8 6 wt (K1R) (K1R) LRR LRR CARD Mscv2.

5 SULEMENTARY INORMATION RESEARCH %LDH Relese *** *** *** *** - A + A / Ln-lA %LDH Relese *** *** 1 *** *** - L2 fla + L2 (la+) 8 6 wt (K1R) (K1R) LRR LRR CARD Mscv2.2 Trnsduced Nlrc4-/- Mcrophges wt (K1R) (K1R) LRR LRR CARD Mscv2.2 Trnsduced Nlrc4-/- Mcrophges c B6 Trnsduced Nlrc4-/- Mcrophges B6 Nlrc4-/- B6 Nlrc4-/- Nlrc4-/- wt (K1R) (K1R) LRR LRR CARD Mscv WB: 42 WB: β-ctin Supplementry igure 6. Complementtion of immortlized Nlrc4 -/- mcrophges with wild-type nd mutnt Nlrc4 lleles. () Immortlized Nlrc4 -/- mcrophges were stly trnsduced with retrovirl vectors encoding wild-type lone or A plus Ln-lA. Mcrophge cell deth (± s.d.) ws mesured y LDH relese. Immortlized B6 mcrophges were used s positive control. () Stly trnsduced mcrophges were infected (MOI=2) with Legionell pneumophil (L2) or flgellin-deficient L. pneumophil fla), nd cell deth (± s.d.) ws mesured 4 hours lter y quntifying LDH relese. (c) Expression level of in B6, Nlrc4 -/-, nd stly trnsduced mcrophges, s ecuse it is constitutively ctive nd kills ny trnsduced cells. Dt shown re representtive of t lest three independent experiments. 5

6 RESEARCH SULEMENTARY INORMATION NAI5 Inflmmsome NAI5 lgellin CARD Cspse1 NAI2 Inflmmsome NAI2 rgj CARD Cspse1 Supplementry igure 7. Working model of NAI/ inflmmsome ssemly. NAI proteins re proposed to medite direct interctions with cteril lignds nd re mintined in n utoinhiited stte in the sence of lignd. NAI5 medites detection of flgellin nd NAI2 medites detection of rgj. In the presence of its stimultory lignd (flgellin, ;; or rgj, ), the of the relevnt NAI protein ssocite with lignd, utoinhiition is relesed, nd NAIs oligomerize vi their nucleotide inding domins (s). Although NAIs re shown here s the sole flgellin/rgj receptor, it is possile tht dditionl proteins, including, re involved in lignd inding. The precise rchitecture nd stoichiometry of the NAI5//flgellin complexes wits further crystllogrphic nlysis. or exmple, NAI5 nd my hetero-oligomerize (not shown). The dptor protein ASC is not shown in the figure ut presumly ssocites with ssemled NAI/ complexes nd hs previously een shown to e 6

7 SULEMENTARY INORMATION RESEARCH Ntive AGE NAI NAI2 - NAI G-lA rgj WB : NAI2 SDS-AGE 11 WB : NAI5 WB : G WB : -ctin Supplementry igure 8. NAI1 does not medite inflmmsome ssemly in response to flgellin or rgj. 293T cells were co-trnsfected with vectors encoding, NAI1, NAI2, NAI5, G-lA, nd/or rgj, followed y ntive gel electrophoresis nd western lotting for oligomerized (top pnel). Lystes were seprted y SDS-AGE nd Western lotted for NAI2, NAI5, 7

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION doi:1.138/nture11444 CMKIIN Gold prticles/ mitochondril re ( m ) 4 3 1 CMKIIN mtcmkiin mtcmkiin SERCA ATP synthse HA mitoplsts mtcmkiin CMKIIN cytosolic mtcmkiin CMKIIN 97 kdl 64 kdl 51 kdl 14 kdl 6 kdl