1. Volume 11, Issue 3, Pages 591-604 (March 2003)
Targeted Disruption of Pyrin, the FMF Protein, Causes Heightened Sensitivity to Endotoxin and a Defect in Macrophage Apoptosis 
Jae Jin Chae, Hirsh D. Komarow, Jun Cheng, Geryl Wood, Nina Raben, P. Paul Liu, Daniel L. Kastner 
Molecular Cell 
Volume 11, Issue 3, Pages (March 2003)
DOI: /S (03)00056-X
Copyright © 2003 Cell Press Terms and Conditions

2. Figure 1 Expression of Mouse Pyrin in RPMϕ
(A) Mefv exons are indicated by shaded boxes. An additional 65 bp coding region is indicated with an open box denoted “a,” with dotted lines indicating alternative splicing. Arrowheads represent primers used in RT-PCR, the products of which are shown to the right (RNA). Western blot with anti-N-mpyrin detects two isoforms in mouse RPMϕ (right, Protein).
(B–D) RPMϕ were stimulated as shown for 24 hr (B and D) or for varying periods (C). Western blots of cell lysates were probed with anti-N-mpyrin.
Molecular Cell  , DOI: ( /S (03)00056-X)
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3. Figure 2 Generation of Pyrin-Truncation Mice
(A) The wt Mefv locus (top), gene-targeting construct (middle), and homologous recombinant Mefv locus (bottom) are shown. Exons are depicted as filled boxes. Locations and transcriptional orientation of PGK-neo, HSV-tk, and EGFP are indicated. The screening probe (probe A) and predicted NdeI restriction fragments from wt and mutant alleles detected by Southern analysis are indicated. The 5′- and 3′-arm PCR products are also shown with locations of primers.
(B) Gancyclovir- and G418-resistant ES clones were screened by Southern blot using probe A following NdeI digestion of genomic DNA (top). The 8.8 kb mutant and 6.1 kb wt alleles are indicated.
(C) RPMϕ pooled from two wild-type (+/+), two heterozygous (+/−), and two homozygous (−/−) mice were treated with LPS and IL-4 for 24 hr. Lysates were subjected to Western blotting using anti-N-mpyrin antibody.
(D) Four pyrin-truncation mice and four wt littermates were injected with 2.5 mg/kg body weight of LPS i.p., and body temperatures were monitored with implanted temperature chips. Data represent the mean ± SE of measurements from four mice. Black horizontal bars represent lights-off periods in a 12:12-hr light-dark cycle. Arrowhead indicates time of injection (0 hr). One of three independent experiments.
Molecular Cell  , DOI: ( /S (03)00056-X)
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4. Figure 3 Pyrin Inhibits IL-1β Processing in Macrophages
(A) RPMϕ from three wt (+/+), three heterozygous (+/−), and three homozygous (−/−) pyrin mutant mice were pooled and treated with 1 μg/ml LPS and 12.5 ng/ml IL-4 for 24 hr. Lysates were subjected to Western blotting using anti-mouse IL-1β, which detects both the 34 kDa pro-IL-1β (upper panel) and 17 kDa activated IL-1β (lower panel).
(B) RPMϕ from nine pyrin mutant mice (−/−) and wt littermates (+/+) were pooled and treated with IL-4, LPS, LPS/IL-4, or no stimulus for 24, 48, and 72 hr. Lysates were subjected to Western blot using anti-mouse IL-1β.
(C) Culture media from cells in (B) were collected and assayed for IL-1β by ELISA. Data represent the mean ± SE duplicate measurements from a representative experiment of three.
(D) RAW cells were transduced with retroviral constructs expressing myc-tagged full-length mouse pyrin (mpyrin-myc), N-terminal truncated mouse pyrin (C-term-myc), or C-terminal truncated mouse pyrin (N-term-myc), or GFP-fused C-terminal truncated mouse pyrin (N-term-GFP) depicted in the schematic above (E) (PYD, pyrin domain; B, B-box zinc finger domain; C-C, coiled-coil domain), and with empty vector control. After G418 selection, cells were treated as indicated for 24 hr. Lysates were subjected to Western blot using anti-myc and anti-mouse IL-1β antibodies.
(E) RAW cells were untreated or treated with LPS/IL-4 or LPS/IL-10 for 24 hr. Supernatants were collected and assayed for IL-1β by ELISA. Data represent the mean ± SE duplicate measurements from a representative experiment of three.
(F) U937 cells were transduced with retroviral constructs expressing wt human pyrin (MEFV) and a mutant pyrin found in FMF patients (M694V), and with empty vector. After G418 selection, cells were treated with LPS for 48 hr. Supernatants were collected and assayed for IL-1β by ELISA. Data represent the mean ± SE from three replicate experiments.
Molecular Cell  , DOI: ( /S (03)00056-X)
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5. Figure 4 Pyrin Regulates Caspase-1 through ASC as an Intermediate
(A) RPMϕ from nine wt and nine pyrin mutant mice were combined into pools and cultured without stimulus (top) or treated with 1 μg/ml LPS (middle) or 1 μg/ml LPS/12.5 ng/ml IL-4 (bottom) for the indicated times. Pro-caspase-1 (p45) and cleaved caspase-1 (p10) were detected by Western blotting with p10-specific anti-mouse caspase-1 antibody.
(B) (a–c) PT67 cells transfected with myc-tagged mouse ASC. Labeled with DAPI (blue, a) and anti-myc antibody followed by AlexaFluor 568 anti-mouse IgG (red, b); merged in (c). (d–f) PT67 cells transfected with full-length mouse pyrin. Labeled with DAPI (blue, d) and polyclonal anti-N-mpyrin antibody followed by AlexaFluor 488 anti-rabbit IgG (green, e); merged in (f). (g–j) PT67 cells cotransfected with full-length mouse pyrin and mouse ASC. ASC and pyrin labeled as described in (a) through (c) and (d) through (f), respectively. g, nuclei (blue); h, pyrin (green); i, ASC (red); merged in (j). (k–n) PT67 cells cotransfected with GFP-fused N-terminal mouse pyrin and mouse ASC. ASC and pyrin labeled as described in (a) through (c) and (d) through (f), respectively. k, nuclei (blue); l, GFP-fused N-terminal pyrin (green); m, ASC (red); merged in (n).
(C) PT67 cells were cotransfected as indicated. Lysates were subjected to GST pull-down assay followed by Western blotting (WB) with anti-myc antibody (upper panel). Expression of full-length and truncated mouse pyrin is shown in the lower panel.
(D) PT67 cells were cotransfected with indicated expression plasmids. Lysates were immunoprecipitated (IP) with anti-myc antibody followed by Western blotting with p10-specific anti-mouse caspase-1 antibody (top). Expression of caspase-1 and ASC were monitored in cell lysates by anti-mouse caspase-1 (middle) and anti-myc (bottom) antibody, respectively.
(E) RAW cells were transduced with vector expressing myc-tagged mouse ASC or empty vector. Lysates from untreated and LPS-treated cells (24 hr) were subjected to Western blotting with anti-myc for mouse ASC (upper panel) or anti-mouse caspase-1 (lower two panels).
(F) Retrovirally transduced cells were treated with LPS or LPS/IL-4 for 24 hr, and lysates were subjected to Western blotting with anti-myc (first panel), or anti-mouse IL-1β (second through fifth panels).
(G) PT67 cells were transfected with GST-ASC, caspase-1, pyrin, or empty vector. GST-ASC lysates were mixed with glutathione-Sepharose beads, which were subsequently mixed with lysates from pyrin and empty vector transfections (lane 1), with caspase-1 and empty vector transfections (lane 2), with pyrin and caspase-1 transfections (lane 3), or with empty vector transfection (lane 4). Mixtures were subjected to GST pull-down followed by Western blotting with antibodies to murine pyrin or caspase-1. Expression of mouse pyrin and mouse caspase-1 is monitored in the lower two panels.
Molecular Cell  , DOI: ( /S (03)00056-X)
Copyright © 2003 Cell Press Terms and Conditions

6. Figure 5 Pyrin Mutant Macrophages Exhibit a Defect in Apoptosis
(A) Pooled plastic-adherent RPMϕ from two pyrin mutant mice (−/−), two heterozygotes (+/−), and two wt (+/+) littermates were treated with 12.5 ng/ml IL μg/ml LPS for 24 hr. DNA fragments were visualized on a 1% agarose gel after ligation-mediated PCR. Lane 1, 100 bp DNA ladder. Lowest band is an amplified fragment of a single-copy gene, En-2, representing the amount of genomic DNA in each sample.
(B) Time course of DNA fragmentation for homozygous pyrin mutant RPMϕ and wt controls.
(C) RPMϕ from wt and homozygotes were treated with IL-4 and LPS ± neutralizing anti-mouse IL-1β antibody (0.5 μg/ml) for 48 hr.
(D) RPMϕ from nine pyrin-truncation mice (−/−) and nine wt littermates (+/+) were combined into pools and treated with IL-4 and LPS for the indicated times. Lysates were subjected to Western blot using antibodies specific to mouse caspases-8 (top) or -9 (bottom).
Molecular Cell  , DOI: ( /S (03)00056-X)
Copyright © 2003 Cell Press Terms and Conditions

7. Figure 6
Proposed Role of Pyrin in Regulating ASC-Mediated Caspase-1 Oligomerization
Left: LPS and proinflammatory cytokines induce ASC, which binds pro-caspase-1 through cognate CARD interactions and leads to caspase-1 oligomerization and autocatalysis. Active p10/p20 subunits cleave pro-IL-1β to mature IL-1β, leading to fever and inflammation. Right: LPS and antiinflammatory cytokines induce pyrin, which binds ASC through homotypic PYRIN domain interactions, sequestering ASC and blocking its interaction with caspase-1.
Molecular Cell  , DOI: ( /S (03)00056-X)
Copyright © 2003 Cell Press Terms and Conditions