Invivogen | Date: 2014-06-10
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No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were blinded to allocation of mice for assessment of histopathology and readouts of inflammation. E. coli strains were routinely cultured aerobically at 37 °C in lysogeny broth (LB) and on LB agar plates. B. abortus was cultured in tryptic soy broth or on tryptic soy agar (TSA) plates,. Chlamydia muridarum strain Nigg II was purchased from ATCC (Manassas, VA). Bacteria were cultured in HeLa 229 cells in DMEM supplemented with 10% FBS. Elementary bodies (EBs) were purified by discontinuous density gradient centrifugations as described previously23 and stored at −80 °C. The HEK293 cell line was maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS at 37 °C in a 5% CO atmosphere. HEK293 cells (ATCC CRL-1573) were obtained from ATCC and were grown in a 48-well tissue culture plates in DMEM containing 10% FBS until ~40% of confluency was reached. HEK293 cells were transfected with a total of 250 ng of plasmid DNA per well, consisting of 25 ng of the reporter construct pNF-κB-luc, 25 ng of the normalization vector pTK-LacZ, and 200 ng of the different combinations of mammalian expression vectors carrying the indicated gene (empty control vector, pCMV-HA-VceC5, pCMV-HA-TRAF2DN (this study), hNOD1-3×Flag, hNOD2-3×Flag, pCMV-HA-hRip2, hNOD1DN-3×Flag, hNOD2DN-3×Flag or pCMV-HA-Rip2DN24 and pCMV-myc-CDC42DN25. The dominant-negative form of TRAF2, lacking an amino-terminal RING finger domain26, was PCR amplified from cDNA prepared from HEK293 cells and cloned into the mammalian expression vector pCMV-HA (BD Biosciences Clontech). Forty-eight hours after transfection, cells were lysed either without any treatment, or stimulated with C12-iE-DAP (1,000 ng ml−1, InvivoGen) and MDP (10 μg ml−1, InvivoGen). After five hours of treatment the cells were lysed and analysed for β-galactosidase and luciferase activity (Promega). FuGene HD (Roche) was used as a transfection reagent according to the manufacturer’s instructions. Cell lines were monitored for mycoplasma contamination. Bone-marrow-derived macrophages (BMDMs) were differentiated from bone marrow precursors from femur and tibiae of C57BL/6 mice obtained from The Jackson Laboratory (Bar Harbor, ME), Nod1+/−Nod2+/− (wild-type littermates) and Nod1−/−Nod2−/− (NOD1/NOD2-deficient) mice (generated at UC Davis) as described previously27. For BMDM experiments, 24-well microtitre plates were seeded with macrophages at a concentration of 5 × 105 cells per well in 0.5 ml of RPMI media (Invitrogen, Grand Island, NY) supplemented with 10% FBS and 10 mM l-glutamine (complete RPMI) and incubated for 48 h at 37 °C in 5% CO . BMDMs were stimulated with C12-iE-DAP (1,000 ng ml−1, InvivoGen), MDP (10 μg ml−1, InvivoGen), thapsigargin (1 μM and 10 μM, Sigma-Aldrich), dithiothreitol (DTT) (1 mM, Sigma-Aldrich), and LPS (10 ng ml−1, InvivoGen) with or without pre-treatment (30 min) of the cells with IRE1α kinase inhibitor KIRA6 (1 μM, Calbiochem), IRE1α endonuclease inhibitor STF-083010 (50 μM, Sigma-Aldrich), PERK inhibitor GSK2656157 (500 nM, Calbiochem) and tauroursodeoxycholate TUDCA (200 μM, Sigma-Aldrich) in the presence of 1 ng ml−1 of recombinant mouse IFNγ (BD Bioscience, San Jose, CA). After 24 h of stimulation, samples for ELISA and gene expression analysis were collected as described below. Preparation of the B. abortus wild-type strain 2308 and the ∆vceC mutant inoculum and BMDM infection was performed as previously described27. Approximately 5 × 107 bacteria in 0.5 ml of complete RPMI were added to each well containing 5 × 105 BMDMs. Microtitre plates were centrifuged at 210g for 5 min at room temperature in order to synchronize infection. Cells were incubated for 20 min at 37 °C in 5% CO , and free bacteria were removed by three washes with PBS, and the zero-time-point sample was taken as described below. After the PBS wash, complete RPMI plus 50 mg ml−1 gentamicin and 1 ng ml−1 of recombinant mouse IFNγ (BD Bioscience, San Jose, CA) was added to the cells, and incubated at 37 °C in 5% CO . For cytokine production assays, supernatant for each well was sampled at 24 h after infection. In order to determine bacterial survival, the medium was aspirated at the time point described above, and the BMDMs were lysed with 0.5 ml of 0.5% Tween 20, followed by rinsing each well with 0.5 ml of PBS. Viable bacteria were quantified by serial dilution in sterile PBS and plating on TSA. For gene expression assays, BMDMs were suspended in 0.5 ml of TRI-reagent (Molecular Research Center, Cincinnati) at the time points described above and kept at −80 °C until further use. At least three independent assays were performed with triplicate samples, and the standard error of the mean for each time point was calculated. All mouse experiments were approved by the Institutional Animal Care and Use Committees at the University of California, Davis, and were conducted in accordance with institutional guidelines. Sample sizes were determined based on experience with infection models and were calculated to use the minimum number of animals possible to generate reproducible results. C57BL/6 wild-type mice and Rip2−/− mice (The Jackson Laboratory), Nod1+/−Nod2+/− (wild-type littermates) and Nod1−/−Nod2−/− (NOD1/NOD2-deficient) mice (generated at UC Davis) were injected intraperitoneally (i.p.) with 100 μl of 2.5 mg per kg body weight of thapsigargin (Sigma-Aldrich) at 0 and 24 h, and 4 h after the second injection the mice were euthanized and serum and tissues collected for gene expression analysis and detection of cytokines. Where indicated, mice were treated i.p. at 12 h before the first thapsigargin dose and 12 h before the second thapsigargin dose with the ER stress inhibitor TUDCA (250 mg per kg body weight). Female and male C57BL/6, Nod1+/−Nod2+/−, Nod1−/−Nod2−/− mice, and Rip2−/− mice aged 6–8 weeks, were held in micro-isolator cages with sterile bedding and irradiated feed in a biosafety level 3 laboratory. Groups of five mice were inoculated i.p. with 0.2 ml of PBS containing 5 × 105 CFU of B. abortus 2308 or its isogenic mutant ∆vceC, as previously described28. At 3 days post-infection, mice were euthanized by CO asphyxiation and their serum and spleens were collected aseptically at necropsy. The spleens were homogenized in 2 ml of PBS, and serial dilutions of the homogenate were plated on TSA for enumeration of CFU. Spleen samples were also collected for gene expression analysis as described below. When necessary, mice were treated i.p. at day one and two post-infection with a daily dose of 250 mg per kg body weight of the ER stress inhibitor TUDCA (Sigma-Aldrich), or 10 mg per kg body weight of the IRE1α kinase inhibitor KIRA6 (Calbiochem) or vehicle control. For the placentitis mouse model, C57BL/6, Nod1+/−Nod2+/− and Nod1−/−Nod2−/− mice, aged 8–10 weeks, were held in micro-isolator cages with sterile bedding and irradiated feed in a biosafety level 3 laboratory. Female Nod1+/−Nod2+/− mice were mated with male C57BL/6 mice (control mice) and female Nod1−/−Nod2−/− mice were mated with male Nod1−/−Nod2−/− mice (NOD1/NOD2-deficient), and pregnancy was confirmed by presence of a vaginal plug. At 5 days of gestation, groups of pregnant mice were mock infected or infected i.p. with 1 × 105 CFU of Brucella abortus 2308 or its isogenic mutant ∆vceC (day 0). At 3, 7 and 13 days after infection mice were euthanized by CO asphyxiation and the spleen and placenta of dams were collected aseptically at necropsy. At day 13 after infection (corresponding to day 18 of gestation), viability of pups was evaluated based on the presence of fetal movement and heartbeat, and fetal size and skin colour. Fetuses were scored as viable if they exhibited movement and a heartbeat, visible blood vessels, bright pink skin, and were of normal size for their gestational period. Fetuses were scored as non-viable if fetal movement, heartbeat, and visible blood vessels were absent, skin was pale or opaque, and their size for gestational period or compared to littermates was small, or they showed evidence of fetal reabsorption. Percentage of viability was calculated as [(number viable pups per litter/total number pups per litter) × 100]. At each time point, the placenta samples were collected for bacteriology, gene expression analysis and blinded histopathological analysis (Extended Data Fig. 6d). When indicated, mice were treated i.p. at days 5, 7 and 9 post-infection with a daily dose of 250 mg per kg body weight of the ER stress inhibitor tauroursodeoxycholate TUDCA (Sigma-Aldrich) or vehicle control. RNA was isolated from BMDMs and mouse tissues using Tri-reagent (Molecular Research Center) according to the instructions of the manufacturer. Reverse transcription was performed on 1 μg of DNase-treated RNA with Taqman reverse transcription reagent (Applied Biosystems). For each real-time reaction, 4 μl of cDNA was used combined with primer pairs for mouse Actb, Il6, Hspa5 and Chop. Real time transcription-PCR was performed using Sybr green and an ABI 7900 RT–PCR machine (Applied Biosystems). The fold change in mRNA levels was determined using the comparative threshold cycle (C ) method. Target gene transcription was normalized to the levels of Actb mRNA. Cytokine levels in mouse serum and supernatants of infected BMDMs were measured using either a multiplex cytokine/chemokine assay (Bio-Plex 23-plex mouse cytokine assay; Bio-Rad), or via an enzyme-linked immunosorbent assay (IL-6 ELISA; eBioscience), according to the manufacturer’s instructions. Cytotoxicity was determined by using a LDH release assay in supernatant of BMDMs treated as described above. LDH release assay was performed using a CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega), following manufacturer’s protocol. The percentage of LDH release was calculated as follows: Percentage of LDH release = 100 × (absorbance reading of treated well − absorbance reading of untreated control)/(absorbance reading of maximum LDH release control − absorbance reading untreated control). The kit-provided lysis buffer was used to achieve complete cell lysis and the supernatant from lysis-buffer-treated cells was used to determine maximum LDH release control. HeLa 229 cells (ATCC CCL-2.1) were cultured in 96-well tissue culture plates at a concentration of 4 × 104 cells per well in Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies, Grand Island, NY) supplemented with 10% FBS. HeLa 229 cells were transfected with a total of 125 ng of pCMV-HA-Rip2DN or empty control vector per well. 24 h post-transfection HeLa 229 cells were treated with Dextran to enhance infection efficacy before they were infected with 1.7 × 105 Chlamydia bacteria per well. The plates were centrifuged at 2,000 r.p.m. for 60 min at 37 °C, then incubated for 30 min at 37 °C in 5% CO Supernatant was discarded and replaced with DMEM containing 1 μg ml−1 cyclohexine (Sigma Aldrich) and where indicated, 1 μM KIRA6, 10 μM thapsigargin or 10 μg ml−1 MDP, was added to cultures before incubation at 37 °C in 5% CO for 40 h. For gene expression assays, HeLa 229 cells were suspended in Tri-reagent (Molecular Research Center, Cincinnati) and RNA was isolated. Infection efficiency was confirmed in separate plates by staining Chlamydia-infected HeLa 229 cells with anti-Chlamydia MOMP antibody and counting bacteria under a fluorescent microscope. Four independent assays were performed and the standard error of the mean calculated. BMDMs stimulated where indicated with 10 μM thapsigargin for 24 h were lysed in lysis buffer (4% SDS, 100 mM Tris, 20% glycerol) and 10 μg of protein was analysed by western blot using antibodies raised against rabbit TRAF2 (C192, #4724, Cell Signaling), rabbit HSP90 (E289, #4875, Cell Signaling), mouse SGT1 (ab60728, Abcam) and rabbit α/β-tubulin (#2148, Cell Signaling). For tissue culture experiments, statistical differences were calculated using a paired Student’s t-test. To determine statistical significance in animal experiments, an unpaired Student’s t-test was used. To determine statistical significance of differences in total histopathology scores, a Mann–Whitney U-test was used. A two-tailed P value of <0.05 was considered to be significant.
University Paul Sabatier, Invivogen and Toulouse 1 University Capitole | Date: 2013-02-15
A method for determining the quantity of anti-HLA antibodies of a liquid medium containing antibodies.
All cells were grown in media supplemented with 10% fetal calf serum (FCS) (Sigma), 100 IU ml−1 penicillin/streptomycin (Sigma) and 2 mM l-glutamine (Sigma), and grown in a humidified incubator at 37 °C with 5% CO . HAP1 (ref. 8) cells and K562 cells (American Type Culture Collection (ATCC)) were cultured in complete IMDM media. HT29, U2OS (both obtained from ATCC), Caco-2, A549 (both gifts from L. Popov), HEK-293T (from Thermo Scientific), H1-HeLa (from ATCC), HuH7 (a gift from P. Sarnow), mouse embryonic fibroblasts (a gift from K. Storek) and NIH3T3 (a gift from W. Kaiser) cells were all cultured in complete DMEM media. Raji cells (expressing DC-SIGN) (a gift from E. Harris) were cultured in complete RPMI media. The cell lines have not been authenticated or tested for mycoplasma contamination. All isogenic knockout clones were grown in the same media as parent cell lines. HAP1 cells were used for haploid genetic screens (see later). Purified, titred stocks of AAV serotypes 1, 2, 3B, 5, 6, 8 and 9 were purchased from University of North Carolina Chapel Hill Gene Therapy Center Vector Core. These were all self-complementary AAV vectors encoding a reporter fluorescent gene (either GFP or RFP). Purified titred stocks of AAV9–luciferase were also purchased from this core facility to perform mouse experiments. Adenovirus type 5 vector carrying mCherry (Ad5–RFP) was constructed by cloning mCherry cDNA in the pAd/CMV/V5-DEST gateway vector (Invitrogen) according to the manufacturer’s protocol. The following antibodies were used in this study: mouse polyclonal anti-KIAA0319L (ab105385) and rabbit polyclonal anti-giantin (ab24586) (Abcam); rabbit polyclonal anti-TGN46 antibody (NBP1-49643) (Novus Biologicals); mouse monoclonal anti-GAPDH (GT239) (Genetex); rabbit polyclonal anti-FGFR1 (D8E4) and rabbit IgG2a isotype control (Cell Signaling Technology); mouse monoclonal phycoerythrin-conjugated anti-MET antibody (95106) and phycoerythrin-conjugated mouse IgG1 isotype control (R&D Systems, Inc). A high-affinity F-actin, fluorescently labelled probe (Alexa Fluor-660 phalloidin) was used to visualize the cell interior and periphery (Life Technologies). Cells were seeded at 10,000 cells per well (96-well plate) overnight. They were then infected with AAV at a MOI of 20,000 vg per cell (unless otherwise specified) in complete DMEM. Virus infectivity was determined 24 h after infection by measuring transgene expression (RFP, GFP or luciferase) using flow cytometry or bioluminescence. In the case of wild-type AAV2 infection, HeLa wild-type or AAVRKO cells were seeded overnight, then infected with wild-type AAV2 (MOI: 1,000 vg per cell) in the presence of wild-type Ad5 (helper virus). RNA was collected using the Ambion Cell-to-C kit (Thermo Scientific) 24 h after infection, and the generated cDNA was used to perform quantitative reverse-transcriptase PCR (RT–qPCR). mRNA levels of the AAV2-encoded rep68 gene were measured (as a means to detect viral replication) and normalized to 18S ribosomal RNA. Primers against rep68 cDNA included: 5′-CCAATTACTTGCTCCCCAAA-3′ and 5′-CGTTTACGCTCCGTGAGATT-3′. Primers against 18S rRNA included: 5′-AGAAACGGCTACCACATCCA-3′ and 5′-CACCAGACTTGCCCTCCA-3′. Ad5–RFP was used to infect cells to obtain 50–60% transduction (Fig. 4a), and flow cytometry was used to measure RFP expression. All infections were performed in triplicate, and all data presented are representative of at least two independent experiments. The haploid genetic screen was performed similarly to the protocol described in ref. 8 with minor changes. Briefly, gene-trap virus was used to create a mutagenized HAP1 library. Of this mutagenized library, 100 million cells were infected with AAV2–RFP at a MOI 20,000 of vg per cell. After 48 h, infected cells were sorted by FACS, where RFP-negative cells (approximately 4% of the population) were sorted and grown over a period of 4 days. The resulting sorted cells were then infected again with AAV2 as before, and re-sorted to enrich the RFP-negative (AAV-resistant) population. Thirty-million cells of the resistant population were used for genomic DNA isolation. We performed sequence analysis of gene-trap insertion sites, and the significance of enrichment for each gene in the screen was calculated by comparing how often that gene was mutated and how often the gene carried an insertion in the control data set (owing to random integration). For each gene, a P value was calculated using the one-sided Fisher exact test in R. The P values were corrected for multiple testing according to the Benjamini and Hochberg method (using the R statistical package), to control for false discovery rate26. In the case of KIAA0319L, the P value was lower than the software could report. The numerical value was thus set to 1 × 10−307 (smallest non-zero normalized floating-point number R could report). CRISPR-Cas9 gene editing technology was used to generate isogenic knockout alleles by targeting exonic sequences shared among all protein-coding transcripts of the respective genes as described in ref. 27. The targeted sequences are depicted in Extended Data Table 1, along with the respective mutations. CRISPR sequence targeting oligonucleotides were designed using the Zhang lab CRISPR design tool (http://crispr.mit.edu). Oligonucleotides corresponding to the guide RNA (gRNA) sequences in Extended Data Table 1 were synthesized (Integrated DNA Technologies). gRNA oligonucleotides were directly cloned into Cas9-expressing plasmids pX330 or pX458 (generated by the Zhang lab; obtained from http://www.addgene.org; plasmid 42230 or 48138). Respective cells were transiently transfected with gRNA-encoding plasmids (and GFP-expressing pcDNA vector with gRNA-pX330 plasmids) using Fugene (Promega). After 48 h, GFP-expressing cells were subcloned using the BD InFlux Cell Sorter at the Stanford Shared FACS facility. They were then expanded over 2 weeks and screened genotypically for the mutated allele by extracting genomic DNA from subclones (using the quick DNA universal 96-kit; Zymo Research), amplifying a 500–700 base-pair (bp) region that encompassed the gRNA-targeted site, and sequencing (ElimBio) the resulting PCR product to identify subclones with knockout mutations. The B3GALT6 isogenic knockout clone was generated using TALENs directed against the nucleotide sequence 5′-TGGCCATGCTGGCCTGGCTG-3′, and the reverse complement sequence of 5′-GAGTTCGTGCTCAAGGCGGA-3′ in the only exon of B3GALT6 (transcript ENST00000379198) as described previously28. One day after transfection, cells were selected with blasticidin S (30 μg ml−1, InvivoGen) for 24 h, then stained using anti-heparan sulfate antibody. Cells displaying low staining intensity were subcloned by FACS. To generate the AAVR full-length construct and ΔC-tail, Gibson assembly reaction kit (New England Biolabs) was used to insert the gene of interest into a lentiviral-based vector, pLenti-CMV-Puro-DEST (w118-1) (plasmid 17452), digested with EcoRV to remove the DEST cassette (a gift from E. Campeau)29. AAVR and derived AAVR genes were amplified from a KIAA0319L cDNA clone (clone ID 3843301) (GE Dharmacon), but a single nucleotide polymorphism at position 447 was changed from a ‘T’ to a ‘G’, allowing the sequence to align to the annotated human genome. The following primers were used to generate PCR products from the human KIAA0319L cDNA to be cloned directly into pLent-CMV-Puro-DEST. AAVR full-length: 5′-ATGTGTGGTGGAATTCTGCAGATACCATGGAGAAGAGGCTGGG-3′ and 5′-CGGCCGCCACTGTGCTGGATTTACTTATCGTCGTCATCCTTGTAATCCAGGATCTCCTCCCGC-3′; ΔC-tail: 5′-GACTCTAGTCCAGTGTGGTG-3′ and 5′-CGGCCGCCACTGTGCTGGATTTACTTATCGTCGTCATCCTTGTAATCTCCTTTTTGCCTCTTACAAC-3′. Note that reverse primer was designed to incorporate a C-terminal 1× Flag-tag sequence. To generate the AAVR deletion constructs, two or three PCR products were generated using the AAVR construct (with Flag-tag) as a template. They were then assembled into the pLenti-CMV-Puro-DEST vector using the Gibson assembly reaction. Primers used to amplify the N-terminal fragments for the following constructs were: ΔMANEC: 5′-GACTCTAGTCCAGTGTGGTG-3′ and 5′-CTCACTGGCATCTGTTGAC-3′; ΔPKD1–2: 5′-GACTCTAGTCCAGTGTGGTG-3′ and 5′-CAGTTCCTTTATAACTGGGTATGG-3′; ΔPKD2–3: 5′-GACTCTAGTCCAGTGTGGTG-3′ and 5′-CTTACGGGGCTCTGGC-3′; ΔPKD3–4: 5′-GACTCTAGTCCAGTGTGGTG-3′ and 5′-GTAATCCACAGCTTTG TTCAC-3′; ΔPKD4–5: 5′-GACTCTAGTCCAGTGTGGTG-3′ and 5′-CTTATTGTTTTCAGGTTGCACAAT-3′; miniAAVR: 5′-GACTCTAGTCCAGTGTGGTG-3′ and 5′-CTCACTGGCATCTGTTGAC-3′; middle fragment of miniAAVR: 5′-GTCAACAGATGCCAGTGAGGTATCTGCTGGAGAGAGTGTC-3′ and 5′-CTTATTGTTTTCAGGTTGCACAAT-3′. Primers used to amplify the C-terminal fragments for the following constructs were: ΔMANEC: 5′-GTCAACAGATGCCAGTGAGACACACTCCTCCAATTCCAT-3′ and 5′-ATCCAGAGGTTGATTGTCGAG-3′; ΔPKD1–2: 5′-CCATACCCAGTTATAAAGGAACTGCCCCCTGTGGCCAACG-3′ and 5′-ATCCAGAGGTTGATTGTCGAG-3′; ΔPKD2–3: 5′-GCCAGAGCCCCGTAAGCCTCCTCAGGCAGATGC-3′ and 5′-ATCCAGAGGTTGATTGTCGAG-3′; ΔPKD3–4: 5′-GTGAACAAAGCTGTGGATTACCCACCTATAGCCAAGATAACTG-3′ and 5′-ATCCAGAGGTTGATTGTCGAG-3′; ΔPKD4–5: 5′-ATTGTGCAACCTGAAAACAATAAGAACCTGGTGGAGATCATCTTGGATATC-3′ and 5′-ATCCAGAGGTTGATTGTCGAG-3′; miniAAVR: 5′-ATTGTGCAACCTGAAAACAATAAGTGTGAGTGGAGCGTGTTATATG-3′ and 5′-ATCCAGAGGTTGATTGTCGAG-3′. AAVR PKD domains 1–5 (residues 311–787) were expressed in E. coli using the pMAL expression system (New England Biolabs). A bacmid, created from a pFastBac dual vector containing the cDNA for the KIAA0319L ectodomain fused to a C-terminal influenza haemagglutinin (HA)-tag was a gift from M. van Oers, and obtained with the assistance of M. Waye30. cDNA coding for PKD domains 1–5 was cloned out of the pFastBacDual expression vector and inserted into the pMAL-c5X vector, using 5′-GTATCTGCTGGAGAGAGTGTCCAGATAACC-3′ and 5′-CAGGTTGTTTTTCCTGCAGGTCACCTGGGATCAGGTTTCAC-3′, then expressed in NEBexpress cells (New England Biolabs). This resulted in a fusion protein comprised of a mannose-binding protein (MBP) tag and AAVR PKD domains 1–5 (referred to as: soluble AAVR). MBP was specifically used as an affinity tag for ease of purification. To create AAVR fusion constructs, Ci-MPR-tail, LDLR-tail and PVR-tail, the Gibson assembly reaction was used to fuse amplified miniAAVR without its C-terminal to the C-terminal of the respective proteins, and insert it into the pLenti-CMV-Puro-DEST vector. Primers used for amplification and insertion included: miniAAVR without C-terminal and transmembrane domain for Ci-MPR-tail: 5′-GACTCTAGTCCAGTGTGGTG-3′ and 5′-CTTATTGTTTTCAGGTTGCACAAT-3′; MPR C-terminal and transmembrane: 5′-ATTGTGCAACCTGAAAACAATAAGGCTGTGGGAGCTGTGC-3′ and 5′-CGGCCGCCACTGTGC-3′; miniAAVR without C-terminal and transmembrane domain for LDLR-tail or PVR-tail: 5′-GACTCTAGTCCAGTGTGGTG-3′ and 5′-CTTATTGTTTTCAGGTTGCACAAT-3′; LDLR or PVR C-terminal and transmembrane: 5′-ATTGTGCAACCTGAAAACAATAAG-3′ and 5′-TAAATCCAGCACAGTGGCGGCCG-3′. Lentiviral transduction was used to create stable cell lines expressing a selected gene of interest under a CMV promoter. Using Gibson assembly reaction, the respective genes of interest (see ‘construction of plasmids’ section) were inserted into the pLenti-CMV-Puro-DEST vector, and used as described previously29. Lentivirus was produced using HEK293 cells and used to transduce the respective cell lines overnight. Cells stably expressing the gene of interest were selected by treatment with 1–3 μg ml−1 puromycin over 2 days (InvivoGen). A lentivirus carrying the mCherry (RFP) gene was used as a control for AAVR complementation in AAVRKO cells. All flow cytometry was performed at the Stanford Shared FACS facility. To perform the haploid genetic screen, FACS was carried out on a FACS Aria flow cytometer (BD). To measure virus transgene expression (RFP/GFP) in all other experiments, cells were trypsinized 24 h after infection and a LSRII-UV flow cytometer (BD) was used to detect fluorescent cells. For cell surface staining, cells were trypsinized and washed using FACS buffer (PBS supplemented with 2% FCS, 1 mM EDTA and 0.1% sodium azide). They were subsequently incubated for 40 min at 4 °C with the respective primary antibodies at a 1:50 dilution (see ‘Antibodies’ section), washed, and incubated for a further 40 min at 4 °C with Alexa488- or Alexa594-conjugated secondary antibodies (1:500 dilution; if the primary was not conjugated) (Life Technologies). This was followed by a final wash and resuspension of cells in FACS buffer before reading fluorescence. All data presented are representative of at least two independent experiments. Data were analysed and assembled using FlowJo software (TreeStar Inc). Cell pellets of 2 × 106 cells were lysed with Laemmli SDS sample buffer containing 5% β-mercaptoethanol and boiled for 10 min at 96 °C. Lysates were separated by SDS–PAGE using the Mini-Protean system (Bio-Rad) on 4–15% polyacrylamide gradient gels (Bio-Rad). Proteins were transferred onto polyvinylidene fluoride membranes (Bio-Rad) using the Bio-Rad Transblot protein transfer system in a semi-wet preparation. Membranes were blocked by incubating with PBS containing 5% non-fat milk for 1 h at room temperature. Membranes were subsequently incubated overnight at 4 °C with primary antibodies at a dilution of 1:1000 (anti-KIAA0319L antibody) or 1:2,000 (anti-GAPDH antibody) in blocking buffer. Membranes were washed 3 times for 5 min using wash buffer (PBS with 0.1% Tween-20), and further incubated in horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-mouse and anti-rabbit 1:5,000 in blocking buffer) (GeneTex) for 1 h at room temperature. After another set of three washes, antibody-bound proteins were visualized on film using the West Pico and Extended Duration chemiluminescence peroxide solutions (Thermo Scientific). Cells were seeded overnight at 40,000 cells per well onto LabTekII glass chamber slides (Thermo Scientific). They were washed once with PBS, and either treated or fixed immediately with 4% paraformaldehyde for 15 min. They were washed three times with PBS before being incubated for 1 h at room temperature with primary antibodies against the respective proteins at a dilution of 1:100 (anti-KIAA0319L and anti-TGN46) or 1:200 (anti-giantin) in immunofluorescence blocking buffer (PBS with 3% BSA, 1% saponin and 1% Triton X-100). Cells were then washed three times in PBS, and incubated for a further hour in DAPI stain (1:500) and fluorescently tagged secondary antibodies (Alexa488 anti-mouse and Alexa594 anti-rabbit; Life Technologies) at a dilution of 1:300. Cells were washed a final three times in PBS, and 5 μl of Vectashield (Vector Laboratories Inc) was applied to each slide chamber before a glass cover slip (VWR International) was placed over slide to mount samples. Cells were visualized directly with a Zeiss LSM 700 confocal microscope. Purification of the soluble AAVR was achieved through amylose-based MBP affinity chromatography (GE Healthcare). ELISA plates (Corning Costar) were coated overnight at 4 °C with 50 μl AAV2 virus-like particles at 2.5 μg ml−1 in 100 mM NaHCO (pH 9.6). Plates were then washed with TBST buffer (0.05% Tween-20 in TBS) and blocked with 3% BSA in TBST for 1 h at room temperature. Subsequent washing was followed by incubation with soluble AAVR or MBP control at the indicated concentrations for 2 h at room temperature. Anti-MBP–HRP (1:500, 1 h incubation at room temperature) was used to detect soluble AAVR and MBP controls, requiring no secondary antibody. Samples were developed with 1-Step Ultra TMB-ELISA substrate as per the manufacturer’s instructions (Thermo Scientific) and optical density assayed by microplate reader (Molecular Devices SpectraMax M2e) at 450 nm. Curve fitting was performed in SigmaPlot v12.5 (Systat Software, Inc). All data presented are representative of at least three independent experiments. Surface plasmon resonance analysis was carried out using a BIAcore X instrument (GE Healthcare) and a flow rate of 10 μl min−1 at 20 °C in HBS-P buffer (10 mM HEPES (pH 7.5), 150 nM NaCl and 0.005% surfactant P20). His-tagged soluble AAVR (His-tagged MBP fusion with AAVR PKD domains 1–5) at various concentrations was mixed with His-tagged MBP to a total concentration of 0.2 μM in 10 mM sodium acetate buffer (pH 4.0) and immobilized on a CM5 sensor chip by amide coupling. MBP at 0.2 μM was sufficient to block nonspecific binding to the dextran. For the analysis of binding affinity, all curves were measured in triplicate and were fitted with a Langmuir 1:1 binding model (BIAevaluation software, GE Healthcare). Wild-type HeLa cells were seeded in 96-well plates at 10,000 cells per well overnight. Anti-AAVR antibody (ab105385) or IgG isotype control (both from Abcam) were incubated with cells (at concentrations ranging from 0.5 to 50 μg ml−1 in DMEM media) for 1 h at 4 °C. Cells were then infected with AAV2–luciferase at a MOI of 1,000 vg per cell, and left for 24 h at 37 °C. A luciferase assay kit (E1500, Promega) was used to detect bioluminescence, with measurements being taken on the Promega GloMax luminometer. Notably, the storage buffers of both antibodies did not contain preservatives such as azide that could interfere with the assay. All data presented are representative of two independent experiments. HeLa cells were seeded in 96-well plates at 10,000 cells per well overnight. Purified soluble AAVR or MBP control was then introduced to the medium at the specified concentrations. Cells were transduced with AAV2–GFP at a MOI of 7,500 vg per cell and incubated for 24 h at 37 °C. This was followed by trypsinization and measuring transgene expression by flow cytometry. For immunofluorescence imaging, the concentration of soluble AAVR and MBP controls was 0.1 μM, and transduction was performed using 7,000 vg per cell. At 24 h post-transduction, cells were incubated with 1 μg ml−1 Hoechst stain (Thermo Scientific) in PBS for 10 min at 37 °C, before washing with PBS and subsequent fluorescent imaging (Nikon Eclipse Ti-E). All data presented are representative of two independent experiments. These experiments were performed similarly to Ci-MPR tracking assays, as described in ref. 31. AAVRKO cells with or without overexpression of AAVR or ΔC-tail were incubated at 4 °C with anti-AAVR antibodies (approximately 25 μg ml−1) for 1 h. Cells were then washed three times with PBS and transferred to 37 °C for specific time points (2, 10, 30 and 60 min), at which time they were fixed with 4% paraformaldehyde for 15 min. Following fixation, immunofluorescence staining (as described earlier) was performed to visualize AAVR endocytosis. All data presented are representative of two independent experiments. All the experiments involving animals were conducted in strict accordance with the Institutional Animal Care and Use Committee of Stanford University. Mice were housed in a Stanford University vivarium, accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Mice were housed in irradiated disposable caging (Innovive Inc) with bi-weekly cage changes. Mice were provided with irradiated food and ultraviolet-irradiated acidified water. Health surveillance was performed via trimester testing of dirty bedding CD1 sentinels (Charles River Laboratories). Sentinels were consistently negative for mouse parvovirus, minute virus of mice, mouse hepatitis virus, rotavirus, mouse encephalomyelitis virus, Sendai virus, mouse adenovirus 1 and 2, ectromelia virus, lymphocytic choriomeningitis virus, pneumonia virus of mice, reovirus 3, Mycoplasma pulmonis, and endo- and ectoparasites. No statistical methods were used to predetermine sample size. In our animal study protocol, we state that the number of animals in each experimental group varies, and is based on similar previous study32. Randomization was not used to allocate animals to experimental groups and the investigators were not blinded to allocation during experiments and outcome assessment. TALEN technology was used to create AAVR isogenic knockout FVB mice (purchased from Cyagen Biosciences). TALEN-targeted sequences were 5′-TGGGAGTCAAGCCAAGTC-3′ and 5′-GCCAGGATATTGTTGGCAGA-3′. Two founder males were mated to FVB/NCrl (Charles River Laboratories) females. After three rounds of breeding, wild-type (Aavr+/+), heterozygous (Aavr+/−) and homozygous AAVRKO (Aavr−/−) mice were generated, determined by genotyping. All genotypes (wild-type, heterozygous and knockout) were obtained in the expected Mendelian ratios after breeding. At 5 weeks of age, 10 female and 9 male animals were used to examine the effect of Aavr KO on AAV infection in vivo. Animals from each group (Aavr+/+, n = 7 (2 litter mates and 5 purchased FVB mice); Aavr+/−, n = 4; Aavr−/−, n = 4 and uninfected mice, n = 4) were injected intraperitoneally with 1 × 1011 viral genomes of AAV9–luciferase in 200 μl of PBS. All of the mice recovered from the injection quickly without loss of mobility or interruption of grooming activity. Aavr+/+ and Aavr−/− mice were found to be significantly different in two independent experiments. The mice were anaesthetized with 2% isofluorane and oxygen. The d-luciferin substrate (Biotium) was injected intraperitoneally (3.3 μg per mouse). After 10 min, the mice were then placed in a light-tight chamber, and images were generated using a cryogenically cooled charge-coupling device camera IVIS 100 (Xenogen), recording bioluminescence at 1, 10, 60 and 100 s. The visual output represents the average radiance as the number of photons emitted per second per cm2 as a false colour image where the maximum is red and the minimum is dark blue. All animals were imaged on a schedule of 3, 7, 10 and 14 days after AAV vector injection. At each time-point a ‘region of interest’ was designated surrounding each animal in order to quantify the radiance (photons s−1 cm−2 sr−1) being released by luciferase activity. This region was kept the same for each mouse and at each time point. The mean and standard deviation of radiance measurements were determined for each mouse group at each time point. The unpaired parametric two-sided Student’s t-test was used for statistical calculations involving two group comparisons in all tissue-culture-based experiments (*P < 0.05, **P < 0.01, ***P < 0.001), with a Welch post-correction accounting for different standard deviations. An unpaired two-sided Mann–Whitney t-test was used for statistical calculations involving two group comparisons in in vivo experiments. GraphPad Prism was used for statistical calculations.
No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. Complementary DNA (cDNA) for human GSDMD was amplified from reverse-transcribed cDNA of HT-29 cells; cDNAs for human GSDMB, human GSDMC and mouse Gsdma3 were synthesized by our in-house gene synthesis facility; cDNAs for human GSDMA and mouse Gsdmd were obtained from Vigene Biosciences (CH892815) and OriGene (MC202215), respectively. The gasdermin cDNAs were inserted into a modified pCS2-3×Flag vector for transient expression in 293T cells and the pWPI lentiviral vector with an N-terminal 2×Flag–HA tag or the FUIGW vector with an N-terminal Flag tag for stable expression in HeLa and iBMDM cells. For recombinant expression in E. coli, the cDNAs were cloned into a modified pET vector with an N-terminal SUMO tag. Truncation mutants of the gasdermins were constructed by the standard PCR cloning strategy and inserted into the pCS2 vector with indicated tags. Expression plasmids for caspase-1, 4, 5 and 11 were previously described4, 9, the caspase-9 plasmid was a gift from X. Wang (National Institute of Biological Sciences, Beijing). cDNAs for human CASP2 and mouse Casp8 are from the Life Technologies Ultimate ORF collection and OriGene (MC200404), respectively. Point mutations were generated by the QuickChange Site-Directed Mutagenesis Kit (Stratagene). All plasmids were verified by DNA sequencing. Antibodies for caspase-1 p10 (sc-515), Myc epitope (sc-789) and GSDMD (sc-81868) were obtained from Santa Cruz Biotechnology. Other antibodies used in this study include anti-HA (MMS-101P, Covance), anti-Flag M2 (F4049), anti-actin (A2066) and anti-tubulin (T5168) (Sigma-Aldrich), rat monoclonal caspase-11 17D9 (NB120-10454, Novus Biologicals), anti-caspase-3 (#9662) and caspase-7 (#12827) (Cell Signaling Technology), IL-1β (3ZD; Biological Resources Branch, National Cancer Institute) and the antibody for detecting endogenous GSDMD (NBP2-33422, Novus Biologicals). Ultrapure LPS from E. coli O111:B4 and poly(dA:dT) were purchased from InvivoGen. LPS (L4524, for priming), TNFα and cycloheximide were purchased from Sigma-Aldrich. SMAC mimetic and the pan-caspase inhibitor zVAD are gifts from the laboratory of X. Wang (National Institute of Biological Sciences, Beijing). Nigericin was purchased from Calbiochem. Recombinant p20/p10 active caspase proteins (caspase-1/2/4/8/9) and lipid A (ALX-581-200-L001) were obtained from Enzo Life Sciences. Cell culture products are from Life technologies and all other chemicals used are Sigma-Aldrich products unless noted. HeLa, HT-29 and 293T cells were obtained from ATCC. C57BL/6 mice-derived wild-type and Tlr4−/−iBMDM cells were kindly provided by K. A. Fitzgerald (University of Massachusetts Medical School, United States) and A. Ding (Weill Cornell Medical College, United States), respectively, and used in our previous studies4, 6, 30. All the cell lines are well-established, commonly used and frequently checked by virtue of their morphological features and functionalities, but have not been subjected to authentication by short tandem repeat (STR) profiling. All the cell lines have been tested to be mycoplasma-negative by the commonly used PCR method. iBMDM, HeLa and 293T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM); HT-29 cells were grown in McCoy’s 5a modified medium. All media were supplemented with 10% (vol/vol) fetal bovine serum (FBS) and 2 mM l-glutamine. All cells were grown at 37 °C in a 5% CO incubator. Transient transfection of HeLa and 293T cells was performed using the JetPRIME (Polyplus Transfection) or Vigofect (Vigorous) reagents by following the manufacturers’ instructions. For stable expression, lentiviral plasmids harbouring the desired gene were first transfected into 293T cells together with the packing plasmids pSPAX2 and pMD2G with a ratio of 5:3:2. The supernatants were collected 48 h after transfection and used to infect HeLa or iBMDM cells for another 48 h. GFP-positive infected cells were sorted by flow cytometry (BD Biosciences FACSAria II). For siRNA knockdown, 0.5 μl of 20 μM siRNA together with 0.8 μl of INTERFERin reagents (Polyplus Transfection) were used for reverse transfection of iBMDM cells in the 96-well plate format; 5 μl of 20 μM siRNA and 10 μl of INTERFERin reagents were used to transfect HeLa cells in the 6-well plate format. The knockdown was performed for 60 h before subsequent analyses. The knockdown efficiency was assessed by quantitative real-time PCR (qRT–PCR) analyses as previously described4. All siRNA oligonucleotides were synthesized by our in-house facility using the sequences from the MISSION shRNA library (Broad Institute, United States) and their sequences are listed in Supplementary Table 1. Activation of the canonical caspase-1 inflammasomes (the NLRP3, NAIP–NLRC4 and AIM2 inflammasomes) and the non-canonical caspase-11 inflammasome by LPS was performed using the protocols that have been detailed in our previous publications4, 6, 9, 20. For bacteria-induced inflammasome activation, S. typhimurium (wild-type and ΔsipD), B. thailandensis (wild-type and ΔbipB), EPEC (wild-type and ΔescN) were used to infect iBMDM cells and S. typhimurium (wild-type and ΔsifA) was used to infect HeLa cells, as described previously4, 9. To examine cell death morphology, cells were treated as indicated in the 6-well plates (Nunc products, Thermo Fisher Scientific Inc.) for static image capture or in glass-bottom culture dishes (MatTek Corporation) for live imaging. Static bright field images of pyroptotic cells were captured using an Olympus IX71 or a Zeiss Pascal Confocal microscope. The image pictures were processed using ImageJ or the LSM Image Examiner program. Live images of cell death were recorded with the PerkinElmer UltraVIEW spinning disk confocal microscopy and processed in the software Volocity. All image data shown were representative of at least three randomly selected fields. The lentiviral gRNA plasmid library for genome-wide CRISPR-Cas9 screen was obtained from Addgene (#50947)33; amplification of the library and preparation of the lentivirus were performed following the protocol provided by Addgene. In brief, 1 μl of library DNA (10 ng μl−1) was used to transform 25 μl of electrocompetent E. coli (TaKaRa). Transformed colonies (>6 × 107) were scraped off the Luria-Bertani (LB) plates into the media, and plasmids were exacted by using the GoldHi EndoFree Plasmid Maxi Kit (CWBIO). To prepare the virus library, 293T cells in the 15-cm dish were transfected with 25 μg of library DNA together with 15 μg of psPAX2 and 10 μg of pMD2.G. Eight hours after transfection, the media were changed to high-serum DMEM (20% FBS with 25 mM HEPES). Another 40 h later, the media (from twenty 15-cm dishes of transfected cells) were collected and centrifuged at 3,000 r.p.m. for 10 min. The supernatant was filtered through a 0.22-μm membrane and aliquots of 30 ml were stored at −80 °C. In the pilot experiment, the volume of the lentivirus library required for achieving an MOI of 0.3 for infecting the target cell line was determined in the 12-well plate format. For the large scale screen, Tlr4−/− iBMDM cells stably expressing the Cas9 protein were seeded in the 15-cm dish (2 × 106 cells in 20 ml media per dish) and a total of 2 × 107 cells were infected with the gRNA lentivirus library. Sixty hours after infection, cells were re-seeded at a density of 1 × 105 ml−1 in fresh media supplemented with 5 μg ml−1puromycin (to eliminate non-infected cells). After 6 to 8 days, ~3 × 108 cells from five culture dishes were electroporated with LPS to trigger caspase-11-mediated pyroptosis9, or stimulated with LFn–BsaK/protective antigen (PA) to induce caspase-1-mediated pyroptosis4; another 3 × 108 cells were left untreated as the control sample. Each screen was repeated another time. Surviving cells were collected after growing to near 90% confluence and lysed in the SNET buffer (20 mM Tris-HCL(pH 8.0), 5 mM EDTA, 400 mM NaCl, 400 µg ml−1 Proteinase K and 1% SDS). Genomic DNAs of each group of cells were prepared by using the phenol-chloroform extraction and isopropanol precipitation method. The DNA was dissolved in H O (4–5 μg μl−1) and used as the templates for amplification of the gRNA. The gRNAs were amplified by a two-step PCR method using the Titanium Taq DNA polymerase (Clontech Laboratories). In the first step, six 50-μl PCR reactions (each containing 50 μg of genomic DNA template) were performed with the forward primer 50bp-F and the reverse primer 50bp-R; the PCR program used is 94 ° C for 180 s, 16 cycles of 94 ° C for 30 s, 60 ° C for 10 s and 72 ° C for 25 s, and a final 2-min extension at 68 ° C. Products of the first-step PCR were pooled together and used as the template for the second-step PCR. Also six 50-μl PCR reactions (each containing 1 μl of the first-step PCR product) were performed with the forward primer Index-F and one of the reverse primers (Index-R1 to R6): Index-R1 for the control sample, Index-R2 for the replicate control sample, Index-R3 for the caspase-11 screen, Index-R4 for the replicate caspase-11 screen, Index-R5 for the caspase-1 screen and Index-R6 for the replicate caspase-1 screen. The PCR program used is 94 ° C for 180 s, 18 cycles of 94 ° C for 30 s, 54 ° C for 10 s and 72 ° C for 18 s, and a final 2-min extension at 68 ° C. Products of the second-step PCR reactions were subjected to electrophoresis on the 1.5% agarose gel; the DNAs (the 310-bp band) were extracted and sequenced at the HiSeq2500 instrument (Illumina) by using the 50-bp single-end sequencing protocol. The first 19 nucleotides from each sequencing read are the gRNA sequence recovered from the library. The frequency of each gRNA was obtained by dividing the gRNA read number by the total sample read number; the fold of enrichment was calculated by comparing the frequency of each gRNA in the experiment sample with that in the control sample. Sequences for all the primers are listed in Supplementary Table 1. The top 50 gRNA hits from the caspase-11 screen were examined and 18 genes that are conserved in human and mouse were identified for siRNA knockdown validation in HeLa cells. HeLa cells expressed caspase-4 but not caspase-5 (Extended Data Fig. 1b) and respond robustly to cytosolic LPS9, 10. For each gene, a mixture of two independent siRNAs was used and the knockdown efficiency of 12 of those having mRNA expression in HeLa cells was confirmed. Importantly, only siRNAs targeting human GSDMD, besides the control CASP4-targeting siRNA, could efficiently block cytosolic LPS-induced pyroptosis (Extended Data Fig. 1c). When assayed individually, the two GSDMD-targeting siRNAs both showed potent inhibition of HeLa cell pyroptosis (Extended Data Fig. 1d). Human codon-optimized Cas9 (hCas9) and GFP-targeting gRNA-expressing plasmids (gRNA_GFP-T1) were purchased from Addgene. The 19-bp GFP-targeting sequence in the gRNA vector was replaced with the sequence targeting the desired gene by QuickChange site-directed mutagenesis. The target sequences used are AGCATCCTGGCATTCCGAG for mouse Gsdmd and TTCCACTTCTACGATGCCA for human GSDMD. To construct the knockout cell lines, 1 μg of gRNA-expressing plasmid, 3 μg of hCas9 plasmid and 1 μg of pEGFP-C1 vector were co-transfected into 6 × 106 iBMDM or HeLa cells. Three days later, GFP-positive cells were sorted into single clones into the 96-well plate by flow cytometry using the BD Biosciences FACSAria II or the Beckman Coulter MoFlo XDP cell sorter. Single clones were screened by the T7 endonuclease I-cutting assay and the candidate knockout clones were verified by sequencing of the PCR fragments as described previously9. The PCR primers used are listed in Supplementary Table 1. All animal experiments were conducted following the Ministry of Health national guidelines for housing and care of laboratory animals and performed in accordance with institutional regulations after review and approval by the Institutional Animal Care and Use Committee at National Institute of Biological Sciences. The Gsdmd knockout mice were generated by co-microinjection of in vitro-translated Cas9 mRNA and gRNA into the C57BL/6 zygotes. Founders with frameshift mutations were screened with T7E1 assay and validated by DNA sequencing. Founders were intercrossed to generate biallelic Gsdmd−/− mice. The gRNA sequence used to generate the knockout mice is AGCATCCTGGCATTCCGAG. C57BL/6 wild-type mice were from Vital River Laboratory Animal Technology Co. and Casp1/11−/− mice were obtained from the Jackson Laboratory. Ripk3−/− mice were a gift from X. Wang (National Institute of Biological Sciences, Beijing). Primary BMDM cells were prepared from 6-week-old male mice (C57BL/6 background) by following a standard procedure as previously described6. For each experimental design, at least two mice were chosen to prepare the BMDM cells for assaying the inflammasome responses; the mice were not randomized and the investigators were not blinded. Relevant cells were treated as indicated. Cell death was measured by the LDH assay using CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega). Cell viability was determined by the CellTiter-Glo Luminescent Cell Viability Assay (Promega). To measure IL-1β release, primary BMDM cells were primed with LPS (1 μg ml−1) for 2 h and released mature IL-1β was determined by using the IL-1β ELISA kit (Neobioscience Technology Company). To obtain recombinant human GSDMD, E. coli BL21 (DE3) cells harbouring pET28a-His -SUMO-GSDMD were grown in LB medium supplemented with 30 μg ml−1 kanamycin. Protein expression was induced overnight at 18 ° C with 0.4 mM isopropyl-B-d-thiogalactopyranoside (IPTG) after OD reached 0.8. Cells were harvested and resuspended in a lysis buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 20 mM imidazole and 10 mM 2-mercaptoethanol. The His -SUMO-tagged protein was first purified by affinity chromatography using Ni-NTA beads (Qiagen) and the SUMO tag was removed by overnight ULP1 protease digestion at 4 °C. The cleaved GSDMD was further purified by HiTrap Q ion-exchange and Superdex G200 gel-filtration chromatography (GE Healthcare Life Sciences). To obtain the constitutive-active caspase-11 p20/p10 tetramer, cDNAs encoding the p20 large and p10 small subunit were cloned into pET21a with a 6×His tag fused to the C terminus of the p10 subunit. The two subunits were separately expressed in E. coli with 1 mM IPTG induction for 4 h at 30 ° C. Bacteria collected from 1-l culture were resuspended and lysed in 100 ml of lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl and 10 mM 2-mercaptoethanol) by sonication. Inclusion bodies, obtained by centrifugation of the lysates at 18,000 r.p.m. for 1 h, was washed with 50 ml of Buffer 1 (50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 M guanidinium hydrochloride (GdnCl) and 0.1% Triton X-100) and 50 ml of Buffer 2 (50 mM Tris-HCl (pH 8.0), 300 mM NaCl and 1 M GdnCl) twice for each buffer. The washed inclusion bodies were solubilized by stirring in 6 ml of the solubilization buffer containing 6.5 M GdnCl, 25 mM Tris-HCl (pH 7.5), 5 mM EDTA and 100 mM DTT overnight at room temperature. To obtain active p20/p10 tetramers by refolding, 12 ml of above solubilized inclusion body solution containing denatured large and small subunits (molecular ratio, 1:2) were drop-by-drop diluted in 500 ml of refolding buffer (100 mM HEPES, 100 mM NaCl, 100 mM sodium malonate, 20% sucrose, 0.1 M NDSB-201 and 10 mM DTT) and then gently stirred in a nitrogen atmosphere at 16 °C overnight. Protein aggregates were removed by centrifugation at 4,000 r.p.m. for 20 min and the refolded protein supernatants were concentrated and dialysed against a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl and 10 mM 2-mercaptoethanol. The protein was affinity-purified by the Ni-NTA beads and further purified by the Superdex G200 gel-filtration chromatography. Expression and purification of recombinant LFn–BsaK and LFn–FliC proteins were described previously4. Recombinant full-length caspase-4 and caspase-11 were expressed and purified from insect cells also as previously described9. Recombinant PreScission protease (PPase) proteins are routine lab stocks. For cleavage by the p20/p10 tetramers of active caspase, 5 μg of purified recombinant GSDMD was incubated with 1 unit of caspase-1, 2, 4, 8 and 9 or 0.1 μg of caspase-11 in a 25-μl reaction containing 50 mM HEPES (pH 7.5), 3 mM EDTA, 150 mM NaCl, 0.005% (vol/vol) Tween-20 and 10 mM DTT. The reaction was incubated for 60 min at 37 ° C. For cleavage by LPS-activated caspase-4/11, the full-length caspase proteins purified from insect cells were first incubated with LPS, lipid A or MDP for 30 min at 30 ° C; 5 μg of purified recombinant GSDMD was then reacted with the ligand-incubated caspases at 37 ° C for 9 min. Cleavage of GSDMD was examined by Coomassie blue staining of the reaction samples separated on the SDS–PAGE gel.