Journal: Nature Communications

Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis

doi: 10.1038/s41467-017-02351-0

Figure Lengend Snippet: SHP2 deficiency results in excessive activation of NLRP3 inflammsome in macrophages. Two groups of macrophages, i.e., peritoneal macrophages from conditional SHP2 knockout (cSHP2-KO) and wild-type (WT) mice a–c , or PMA-differentiated THP-1 cells with shRNA-Ctrl or shRNA-SHP2 lentivirus d , f , were primed with 100 ng ml −1 LPS for 3 h, and then stimulated with ATP (5 mM, 1 h), MSU (500 µg ml −1 , 2 h) or Nigericin (10 µM, 2 h), respectively. a , b , d , e Enzyme-linked immunosorbent assay (ELISA) of IL-1β and IL-18 in culture supernatants. ND represents not detectable. c , f Immunoblot analysis of cell lysates. g ELISA of IL-1β in supernatants of THP-1-derived macrophages left untreated or treated with SHP2 inhibitor PHPS1 (10 μM) or NSC87877 (10 μM) for 1 h, followed by LPS treatment and ATP, MSU or Nigericin stimulation. h Immunoblot analysis of proteins immunoprecipitated with anti-ASC from lysates of ATP-treated SHP2 knockdown THP-1-derived macrophages. i Immunoblot analysis of ASC in cross-linked pellets (upper panels) and cell lysates (lower panels) from ATP-treated SHP2 knockdown THP-1-derived macrophages. * P < 0.05, ** P < 0.01, one-way ANOVA for multiple comparisons. Data are representative of three independent experiments (mean and SEM of three independent samples in a , b , d , e , g )

Article Snippet: pET21b-caspase-1-His (Plasmid 11809), pCI-ASC-HA (Plasmid 41553), pGEX-4T1 SHP2 WT (Plasmid 8322), and pCMV-SHP2 (Plasmid 8381) were purchased from Addgene.

Techniques: Activation Assay, Knock-Out, shRNA, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Immunoprecipitation, Knockdown

Journal: Nature Communications

Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis

doi: 10.1038/s41467-017-02351-0

Figure Lengend Snippet: SHP2 deficiency in macrophages aggravates murine peritonitis model. a – c Eight-week-old female cSHP2-KO and WT mice were killed 12 h after Alum injection and peritoneal cavities were washed with PBS. a Flow cytometry analysis of peritoneal exudate cells (PECs) 12 h after Alum injection in mice. b ELISA of IL-1β level in the lavage fluid 8 h after Alum injection in mice. c Immunoblot analysis (left panel) and flow cytometry analysis (right panel) of caspase-1 activation in PECs 12 h after Alum injection in mice. Data are representative of three independent experiments (mean and SEM of 10 mice per group), * P < 0.05 by Student’s t -test

Article Snippet: pET21b-caspase-1-His (Plasmid 11809), pCI-ASC-HA (Plasmid 41553), pGEX-4T1 SHP2 WT (Plasmid 8322), and pCMV-SHP2 (Plasmid 8381) were purchased from Addgene.

Techniques: Injection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay

Journal: Nature Communications

Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis

doi: 10.1038/s41467-017-02351-0

Figure Lengend Snippet: SHP2 deficiency leads to mitochondrial dysfunction and excessive NLRP3 inflammasome activation. a , b Flow cytometry analysis of mitochondrial membrane potential by JC-1 staining a or mitochondrial ROS by MitoSOX staining b in peritoneal macrophages from conditional SHP2 knockout (cSHP2-KO) and wild-type (WT) mice, and left untreated or treated with ATP (5 mM) for indicated times. c Quantitative real-time PCR analysis of mtDNA released from peritoneal macrophages from cSHP2-KO and WT mice and left unstimulated (medium) or primed with LPS (100 ng ml −1 ) for 3 h and stimulated with ATP (5 mM, 1 h) and Nigericin (10 µM, 2 h). d ELISA of IL-1β in supernatants of peritoneal macrophages from cSHP2-KO and WT mice, which were primed with LPS (100 ng ml −1 ) for 3 h, and left untreated or treated with Ac-YVAD-cmk (30 μM), NAC (5 mM), or E-64d (20 μM) for 1 h, followed by stimulation of ATP (5 mM) for 1 h. * P < 0.05, ** P < 0.01, one-way ANOVA for multiple comparisons; NS represents no significance. Data are presented as mean ± SEM of three independent experiments in a – d

Article Snippet: pET21b-caspase-1-His (Plasmid 11809), pCI-ASC-HA (Plasmid 41553), pGEX-4T1 SHP2 WT (Plasmid 8322), and pCMV-SHP2 (Plasmid 8381) were purchased from Addgene.

Techniques: Activation Assay, Flow Cytometry, Membrane, Staining, Knock-Out, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis

doi: 10.1038/s41467-017-02351-0

Figure Lengend Snippet: SHP2 translocates into mitochondrial matrix during NLRP3 inflammasome activation. a Immunofluorescence analysis of SHP2 and mitochondria from bone marrow-derived macrophages with untreated (medium) or ATP (5 mM, 15 min), or MSU (500 µg ml −1 , 2 h) or Nigericin (10 µM, 2 h) treatment. Scale bar, 5 µm. b Immunoblot analysis of mitochondrial and cytosolic components of THP-1-derived macrophages treated with ATP (5 mM) for indicated times. c Immunoblot analysis of SHP2 location in mitochondria from THP-1-derived macrophages. Cells were treated with 5 mM ATP for 30 min, then mitochondria were isolated and incubated with 40 μM proteinase K for 30 min. Tom20 in mitochondrial outer membrane (MOM) and Tim23 in mitochondrial inner membrane (MIM) were used as controls, respectively. d Immunoblot analysis of SHP2 expression in submitochondrial fractions from THP-1-derived macrophages treated with ATP (5 mM, 30 min). Tom20, COX IV, and HSP60 were used to represent MOM, MIM, and mitochondrial matrix protein, respectively. e Immunofluorescence analysis of SHP2 and Tom20 from bone marrow-derived macrophages with untreated (medium) or ATP (5 mM, 30 min) by structured-illumination microscopy (SIM). Scale bar, 5 µm. Data are representative of three independent experiments

Article Snippet: pET21b-caspase-1-His (Plasmid 11809), pCI-ASC-HA (Plasmid 41553), pGEX-4T1 SHP2 WT (Plasmid 8322), and pCMV-SHP2 (Plasmid 8381) were purchased from Addgene.

Techniques: Activation Assay, Immunofluorescence, Derivative Assay, Western Blot, Isolation, Incubation, Membrane, Expressing, Microscopy

Journal: Nature Communications

Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis

doi: 10.1038/s41467-017-02351-0

Figure Lengend Snippet: SHP2 interacts with ANT1 during NLRP3 inflammasome activation. a Sliver staining of GST pull-down proteins using GST-SHP2 fusion protein. The proteins interacting with GST-SHP2 were identified by mass spectrometry. b Immunofluorescence analysis of SHP2 and ANT1 from bone marrow-derived macrophages with untreated (medium) or ATP (5 mM, 15 min) treatment. Scale bar, 5 µm. c Immunoblot analysis of reciprocal co-immunoprecipitation (Co-IP) from THP-1-derived macrophages treated with ATP (5 mM) for indicated times. d Immunoblot analysis of Co-IP from mitochondrial and cytosolic components in THP-1-derived macrophages treated with ATP for 15 min. e Immunoblot analysis of reciprocal Co-IP from HEK293T cells overexpressing HA-tagged SHP2 and myc-tagged ANT1. Data are representative of three independent experiments

Article Snippet: pET21b-caspase-1-His (Plasmid 11809), pCI-ASC-HA (Plasmid 41553), pGEX-4T1 SHP2 WT (Plasmid 8322), and pCMV-SHP2 (Plasmid 8381) were purchased from Addgene.

Techniques: Activation Assay, Staining, Mass Spectrometry, Immunofluorescence, Derivative Assay, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis

doi: 10.1038/s41467-017-02351-0

Figure Lengend Snippet: Tom20/Tom40 and Tim23 complex are necessary for SHP2 translocation to mitochondrial matrix. a Predication of mitochondrial target sequence in SHP2 by PSORT II. b Immunoblot analysis of GFP localization in submitochondrial fractions from HEK293T cells which were transfected with GFP or RRWFH-GFP plasmid. c Immunoblot analysis of SHP2 localization in submitochondrial fractions from HEK293T cells which were transfected with SHP2-HA or SHP2-mut-HA (mitochondrial target sequence mutation, RRWFH mutated to AAWFH) plasmid followed by ATP treatment (5 mM, 30 min). d Immunofluorescence analysis mitochondrial localization of GFP-tagged RRWFH motif in HEK293T cells. Scale bar, 10 µm. e Immunofluorescence analysis mitochondrial localization of SHP2-HA or SHP2-mut-HA plasmid in HEK293T cells. Scale bar, 10 µm. f Co-immunoprecipitation (Co-IP) analysis of the interaction of SHP2 and ANT1 in HEK293T cells, which were transfected with ANT1-myc and SHP2-HA or SHP2-mut-HA. g Flow cytometry analysis of mitochondrial membrane potential by JC-1 staining in HEK293T cells which were transfected with SHP2-HA or SHP2-mut-HA plasmid followed by cccp treatment (20 µM, 1 h). h Immunoblot analysis of SHP2 in mitochondria after Tom20 or Tom40, or Tom70 sliencing. Endogenous Toms were separately knocked down by its corresponding shRNAs in THP-1 cells followed by ATP treatment (5 mM, 30 min), then mitochondria were isolated and incubated with 40 μM proteinase K (Pro K) for 30 min. Tom20 in mitochondrial outer membrane (MOM) and Tim23 in mitochondrial inner membrane (MIM) were used as control, respectively. i Immunoblot analysis of SHP2 expression in submitochondrial fractions from THP-1-derived macrophages treated with ATP (5 mM, 30 min) after Tim22 or Tim23 silencing. * P < 0.05 by Student’s t -test, NS represents no significance. Data are representative of three independent experiments (mean and SEM of three independent samples in g )

Article Snippet: pET21b-caspase-1-His (Plasmid 11809), pCI-ASC-HA (Plasmid 41553), pGEX-4T1 SHP2 WT (Plasmid 8322), and pCMV-SHP2 (Plasmid 8381) were purchased from Addgene.

Techniques: Translocation Assay, Sequencing, Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Immunofluorescence, Immunoprecipitation, Co-Immunoprecipitation Assay, Flow Cytometry, Membrane, Staining, Isolation, Incubation, Control, Expressing, Derivative Assay

Journal: Nature Communications

Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis

doi: 10.1038/s41467-017-02351-0

Figure Lengend Snippet: SHP2 inhibits NLRP3 inflammasome activation in an ANT1-dependent manner. a , b SHP2 knockdown, ANT1 knockdown and SHP2-ANT1 double knockdown THP-1-derived macrophages were primed with 100 ng ml −1 LPS for 3 h, followed by ATP (5 mM, 1 h), MSU (500 µg ml −1 , 2 h), or Nigericin (10 µM, 2 h) stimulation, respectively. a ELISA of IL-1β in the supernatant. b Quantitative real-time PCR analysis of mtDNA. c ELISA of IL-1β in the culture supernatant from ANT1 knockdown THP-1-derived macrophages and left untreated or treated with NSC87877 (10 μM) or PHPS1 (10 μM) for 1 h, followed by ATP or Nigericin stimulation. d – f LPS-primed SHP2 knockdown THP-1-derived macrophages were treated with CATR (5 mM) or BA (50 µM) for 1 h, followed by ATP or Nigericin stimulation. d Immunoblot analysis of Co-IP from THP-1-derived macrophages treated with CATR or BA. e ELISA of IL-1β in the culture supernatant. f Immunoblot analysis of cell lysates from THP-1-derived macrophages treated with CATR or BA. * P < 0.05, ** P < 0.01, one-way ANOVA for multiple comparisons, NS represents no significance. Data are representative of three independent experiments (mean and SEM of three independent samples in a – c , e )

Article Snippet: pET21b-caspase-1-His (Plasmid 11809), pCI-ASC-HA (Plasmid 41553), pGEX-4T1 SHP2 WT (Plasmid 8322), and pCMV-SHP2 (Plasmid 8381) were purchased from Addgene.

Techniques: Activation Assay, Knockdown, Derivative Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Western Blot, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis

doi: 10.1038/s41467-017-02351-0

Figure Lengend Snippet: SHP2 dephosphorylation of ANT1 at Tyr 191 is essential for mitochondrial homeostasis. a Flow cytometry analysis of mitochondrial membrane potential by JC-1 staining of HEK293T cells overexpressing Vector, SHP2-HA, SHP2-D61A-HA, or SHP2-C459S-HA plasmid and left untreated (medium) or treated with cccp (20 µM, 1 h). b , c HEK293T cells were transfected with pro-caspase-1, ASC, NLRP3, and SHP2-HA, SHP2-D61A-HA, or SHP2-C459S-HA plasmid, respectively, followed by ATP (5 mM, 1 h) treatment. b Flow cytometry analysis of caspase-1 activation. c Immunoblot analysis of caspase-1 activation. d Flow cytometry analysis of mitochondrial membrane potential by JC-1 staining of HEK293T cells overexpressing Vector, ANT1-myc, ANT1-Y191F-myc, or ANT1-Y195F-myc plasmid and left untreated (medium) or treated with cccp (20 µM, 1 h). e , f HEK293T cells were transfected with pro-caspase-1, ASC, NLRP3, and ANT1-myc, ANT1-Y191F-myc or ANT1-Y195F-myc plasmid respectively followed by ATP (5 mM, 1 h) treatment. e Flow cytometry analysis of caspase-1 activation. f Immunoblot analysis of caspase-1 activation. * P < 0.05, one-way ANOVA for multiple comparisons, NS represents no significance. Data are representative of three independent experiments (mean and SEM of three independent samples in a , b , d , e )

Article Snippet: pET21b-caspase-1-His (Plasmid 11809), pCI-ASC-HA (Plasmid 41553), pGEX-4T1 SHP2 WT (Plasmid 8322), and pCMV-SHP2 (Plasmid 8381) were purchased from Addgene.

Techniques: De-Phosphorylation Assay, Flow Cytometry, Membrane, Staining, Plasmid Preparation, Transfection, Activation Assay, Western Blot

Journal: Nature Communications

Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis

doi: 10.1038/s41467-017-02351-0

Figure Lengend Snippet: The graphic illustration of the mechanism of SHP2 regulating NLRP3 inflammasome activation. Stimulated by Signal 2 activators (e.g., ATP, MSU, and Nigericin), SHP2 is recruited to mitochondria through its RRWFH motif. With the help of Tom20/Tom40 and Tim23 complex, SHP2 translocates into the mitochondrial matrix and dephosphorylates ANT1 at Tyr 191. This serves as a key mechanism controlling mitochondrial homeostasis, preventing leakage of mitochondrial DNA (mtDNA) and overproduction of reactive oxygen species (ROS), which results in negative regulation of NLRP3 inflammasome activation

Article Snippet: pET21b-caspase-1-His (Plasmid 11809), pCI-ASC-HA (Plasmid 41553), pGEX-4T1 SHP2 WT (Plasmid 8322), and pCMV-SHP2 (Plasmid 8381) were purchased from Addgene.

Techniques: Activation Assay