SUPPLEMENTARY INFORMATION doi:10.1038/nature11429 S1a 6 7 8 9 Nlrc4 allele S1b Nlrc4 +/+ Nlrc4 +/F Nlrc4 F/F 9 Targeting construct 422 bp 273 bp FRT-neo-gb-PGK-FRT 3x.STOP S1c Nlrc4 +/+ Nlrc4 F/F casp1 p45 casp1 p20 casp1 p10 LPS + Flagellin LPS + ATP LPS + dsdna - + - - - - + - - - - + - - - - - + - - - - + - - - - + - - - - - + - - - - + - - - - + - - - - - + - - - - + - - - - + IL-1β p17 Supplementary Figure 1. Nlrc4 F/F knock-in mice. (a) Nlrc4 F/F mice were generated from C57BL/6 C2 ES cells after insertion, by homologous recombination, of a 3x sequence (red) into Nlrc4 exon 9 ahead of its translation termination codon. The neo selection cassette was removed from the ES cells with Flp-expressing adenovirus. (b) PCR genotyping of Nlrc4 +/+, Nlrc4 +/F, and Nlrc4 F/F mice. (c) Nlrc4 +/+,, and Nlrc4 F/F BMDMs infected with wildtype strain SL1344 for 4 h, or primed with LPS for 4 h and then stimulated with either ATP or dsdna for 30 min, or transfected flagellin for 4 h. Western blots (WB) analyzed cell lysate combined with culture supernatants. WWW.NATURE.COM/NATURE 1
RESEARCH SUPPLEMENTARY INFORMATION S2a 1 MNFIRNNRRA LIQRMGLTVT KQICDDLFAL NVLNNQEANV IYCEPLEQEA 51 ARKIIHMTMQ KGSAACNLFL KSLENWDYFV YQDLTGQNLS YQVTEEDLSV 101 LAQNLKDLYN SPAFLNFYPL GEDIDIIFNL EKTFTEPIMW KKDHRHHRVE 151 QLTLGSLLEA LKSPCLIEGE SGKGKSTLLQ RIAMLWASGG CRALKGFRLV 201 FFIHLRSARG GLFETLYDQL LNIPDFISKP TFKALLLKLH KEVLFLLDGY 251 NEFHPQNCPE IEALIKENHR FKNMVIVTTT TECLRHIRHV GALTAEVGDM 301 TEDSAKDLIE AVLVPDQVER LWAQIQESRC LRNLMKTPLF VVITCAIQMG 351 RQEFQAHTQT MLFQTFYDLL IQKNSHRYRG GASGNFARSL DYCGDLALEG 401 VFAHKFDFEP EHGSSMNEDV LVTIGLLCKY TAQRLKPTYK FFHKSFQEYT 451 AGRRLSSLLT SKEPEEVSKG NSYLNKMVSI SDITSLYGNL LLYTCGSSTE 501 ATRAVMRHLA MVYQYGSLQG LSVTKRPLWR QESIQSLRNT TEQDVLKAIN 551 VNSFVECGIN LFSESMSKSD LSQEFEAFFQ GKSLYINSEN IPDYLFDFFE 601 YLPNCASALD FVKLDFYERA TESQDKAEEN VPGVHTEGPS ETYIPPRAVS 651 LFFNWKQEFK TLEVTLRDIS KLNKQDIKYL GKIFSSATNL RLHIKRCAAM 701 AGRLSSVLRT CKNMHTLMVE ASPLTTDDEQ YITSVTGLQN LSIHRLHTQQ 751 LPGGLVDSLG NLKNLERLIL DDIRMNEEDA KSLAEGLRSL KKMRLLHLIH 801 LSDIGEGMDY IVKSLSEESC DLQEMKLVAC CLTANSVKVL AQNLHNLIKL 851 SILDISENYL EKDGNEALQE LIGRLGVLGE LTTLMLPWCW DVHTSLPKLL 901 KQLEGTPGLA KLGLKNWRLR DEEIKSLGEF LEMNPLRDLQ QLDLAGHRVS 951 SDGWLYFMNV FENLKQLVFF DFSTEEFLPD AALVRKLSQV LSKLTLLQEV 1001 KLTGWEFDDY DISAIKGTFK LVTA S2b IP HEK 293T WT S533A Supplementary Figure 2. Phosphorylation of NLRC4 Ser533 by mass spectrometry and the generation of an anti-phospho-ser533 NLRC4 () antibody. (a) NLRC4 peptides detected by mass spectrometry covered 84% of the NLRC4 protein sequence. Residues not covered are printed grey. Phosphorylation was detected only on Ser533 (boxed in red) after BMDMs were infected with. (b) 293T cells were transfected with NLRC4. (WT in lane 1, or S533A in lane 2) and the tagged proteins affinity purified with anti- agarose. The proteins were then blotted with phospho-s533 (upper panel) or antibodies (lower panel). 2 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH S3a S3b +WT NLRC4 4000 +S533A NLRC4 +WT NLRC4 +S533A NLRC4 Nlrc4 F/F 3000 IP WB NLRC4 Clone 3C4 Clone 3F8 Clone 4E2 Clone 4B2 IL-6 (pg/ml) 2000 1000 0 - LPS+ATP LPS+dsDNA Supplementary Figure 3. Characterization of NLRC4-reconstituted macrophages. BMDMs were immortalized with ER-Hoxb8, reconstituted with WT NLRC4 or the S533A mutant, single cell cloned, and then differentiated into macrophages. (a) WT or S533A NLRC4 expression in reconstituted macrophages (two independent clones of each genotype) was compared with endogenous NLRC4 expression in Nlrc4 F/F BMDMs by IP anti-/wb anti-nlrc4. (b) IL-6 secretion from macrophages infected with S. typhimurium for 4 h, or stimulated with LPS/ATP or LPS/transfected dsdna for 30 min. Bars show the mean ± s.d. of 3 independent experiments. WWW.NATURE.COM/NATURE 3
RESEARCH SUPPLEMENTARY INFORMATION S4a S4b Superdex 200 Nlrc4 +/+ BMDM Fraction 1 2 3 4 5 6 7 8 9 10 11 Lys infection Cytosolic extract Mono Q Superdex 200 Fraction 1 2 3 4 5 6 7 8 9 10 Lys Mono Q Superdex 200 Superdex 200 Fraction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Lys Mono Q S4c ppkcδ Mono Q Time (min) 0 15 30 60 PKCδ Fraction 1 2 3 4 5 6 7 8 9 10 11 12 MS analysis Lys Supplementary Figure 4. Purification of the NLRC4 kinase. (a) Purification scheme for identifying the NLRC4 kinase. (b) Column fractions were assayed for their ability to phosphorylate affinity purified NLRC4.3x in vitro. Phosphorylation on Ser533 was detected by immunoblotting with a phospho-s533 NLRC4 antibody. (c) Nlrc4 +/+ BMDMs were primed with LPS for 16 h and then infected with. 4 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH S5a S5b 40 IP WB pnlrc4 casp1 p45 LDH% Release 30 20 10 Rottlerin (μm) Gö 6976 (μm) PKC θ inhibitor (μm) 0 5 10 - - - - - - - 10 25 - - - - - - - 10 25 + + + + + + + casp1 p10 IL-1β p17 0 - DMSO Rottlerin (10 μm) Gö 6976 (10 μm) PKC θ inhibitor (10 μm) S5c Merged DAPI DMSO Rottlerin (10 μm) Supplementary Figure 5. Rottlerin abrogates -stimulated NLRC4 phosphorylation, caspase-1 activation, and pyroptosis without affecting bacteria uptake. (a-b) Nlrc4 F/F BMDMs were infected with in the presence of the inhibitors indicated for 4 h. Cells plus medium were analyzed in (a). LDH release in (b) is the mean ± s.d. of 3 independent experiments. (c) Nlrc4 F/F BMDMs were infected with in the presence of DMSO vehicle or 10 µm Rottlerin for 1 h. DNA stained with DAPI is blue; staining is green. Bars, 10 m. WWW.NATURE.COM/NATURE 5
RESEARCH SUPPLEMENTARY INFORMATION S6a Merged ASC Caspase-1 DAPI Prkcd +/+ Prkcd -/- S6b P=0.0332 S6c 60 Prkcd +/+ 25 P=0.0102 Prkcd -/- % cells with an ASC focus 20 15 10 LDH% Release 40 20 P=0.0825 P=0.0072 5 0 Prkcd +/+ Prkcd -/- 0 0 1 2 3 Time (h) S6d Merged DAPI Prkcd +/+ Prkcd -/- 6 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 6. PKCδ deficiency attenuates ASC focus formation in S. typhimurium-infected BMDMs. (a-c) Prkcd +/+,, and Prkcd -/- BMDMs were infected with (MOI 5) for 3 h in the presence of zvad-fmk. DNA stained with DAPI is blue, caspase-1 p10 staining is red, and ASC staining is green; bars, 10 µm. (a). The percentage of cells with an ASC focus was determined by counting at least 400 cells. Graphs represent the mean ± s.d. of 3 experiments. P-values were determined by 2-tailed t-test (b). (c) LDH release from BMDMs infected with (MOI 1). Bars represent the mean ± s.e.m. of 3 independent experiments. P-values were determined by 2-tailed t-test. (d) Prkcd +/+ and Prkcd -/- BMDMs were infected with (MOI 5) for 1 h. DNA stained with DAPI is blue; staining is green. Bars, 10 µm. WWW.NATURE.COM/NATURE 7
RESEARCH SUPPLEMENTARY INFORMATION Supplementary Table 1 GFP + cell % PMX WT S533A S533D 3 days post infection 85% 82% 86% 93% 15 days post sorting GFP + cells 64% <10% 38% <1% Supplementary Table 1. NLRC4 phosphomimetic S533D is deleterious to immortalized macrophage progenitors. The frequency of GFP+ immortalized progenitors at different times after transduction with GFP-expressing retroviruses that lack NLRC4 (PMX) or also express wild-type (WT) NLRC4, NLRC4 S553A, or NLRC4 S553D was determined by flow cytometry. Supplementary Table 2 Description (UniPort) Gene (UniPort) PFam Mol.Wt. Total Peptides Unique Peptides Protein kinase C delta type Prkcd Pkinase C1_1 Pkinase_C 77547 24 15 Serine/ threonine - protein kinase PAK2 Pak2 Pkinase PBD 57930 4 4 Supplementary Table 2. Mass spectrometry identifies two putative NLRC4 kinases: PKC and PAK2. 8 WWW.NATURE.COM/NATURE