Journal: Biochemistry

Article Title: Targeted degradation of the oncogenic phosphatase SHP2

doi: 10.1021/acs.biochem.1c00377

Figure Lengend Snippet: (A) Chemical structure of RMC-4550 and X-ray crystal structure of SHP2 in complex with RMC-4550 (PDB code 7RCT). Surface representation of SHP2 in complex with RMC-4550 bound in the central tunnel formed at the interface of N-SH2 (green), C-SH2 (blue) and PTP (wheat) domains. (B) Chemical structures of RMC-4550-based PROTAC candidates, R1–1C, R1–3C and R1–5C.

Article Snippet: Antibodies used in this study were obtained commercially from the following sources: SHP2 (Bethyl, #A301–544A), Phospho-Thr202/Tyr204-Erk1/2 (CST, #9101), Cereblon (CST, #71810), b-actin (Millipore-Sigma, #A1978), b -tubulin (CST, #2146), GAPDH (CST, #5174).

Techniques:

Journal: Biochemistry

Article Title: Targeted degradation of the oncogenic phosphatase SHP2

doi: 10.1021/acs.biochem.1c00377

Figure Lengend Snippet: (A) Inhibition of SHP2-F285S- or PTP-mediated DIFMUP dephosphorylation by R1–1C, R1–3C, R1–5C and RMC-4550. MV4;11 cells were treated with increasing doses of R1–3C (B), R1–1C or R1–5C (C) for 24 h and subjected to Western blotting using SHP2, GAPDH and β-actin antibodies. Quantification of band intensities on the gels are shown below the blots.

Article Snippet: Antibodies used in this study were obtained commercially from the following sources: SHP2 (Bethyl, #A301–544A), Phospho-Thr202/Tyr204-Erk1/2 (CST, #9101), Cereblon (CST, #71810), b-actin (Millipore-Sigma, #A1978), b -tubulin (CST, #2146), GAPDH (CST, #5174).

Techniques: Inhibition, De-Phosphorylation Assay, Western Blot

Journal: Biochemistry

Article Title: Targeted degradation of the oncogenic phosphatase SHP2

doi: 10.1021/acs.biochem.1c00377

Figure Lengend Snippet: (A) Time course of SHP2 degradation by R1–5C (100 nM) in MV4;11 cells. Immunoblotting with SHP2 and β-actin antibodies. (B) CRBN−/− and parental MOLT4 cells were treated with increasing doses of R1–5C for 24 h and subjected to Western blotting using SHP2, CRBN and β-actin antibodies. Quantification of band intensities on the gels are shown below the blots.

Article Snippet: Antibodies used in this study were obtained commercially from the following sources: SHP2 (Bethyl, #A301–544A), Phospho-Thr202/Tyr204-Erk1/2 (CST, #9101), Cereblon (CST, #71810), b-actin (Millipore-Sigma, #A1978), b -tubulin (CST, #2146), GAPDH (CST, #5174).

Techniques: Western Blot

Journal: Biochemistry

Article Title: Targeted degradation of the oncogenic phosphatase SHP2

doi: 10.1021/acs.biochem.1c00377

Figure Lengend Snippet: (A-D) Scatterplots displaying relative fold-change in SHP2 abundance following treatment of MV4;11 cells with 100 nM R1–5C for 4 h (A), 8 h (B), 16 h (C) or 100 nM RMC-4550 (D). SHP2/PTPN11 is highlighted in red. Hits highlighted in blue in (C) and (D) indicate changes in abundance of proteins at 16 h time point due to secondary effects (such as transcriptional responses) of SHP2 degradation or inhibition. (E) Heatmap of the protein abundance changes in MV4;11 cells comparing treatment with 100 nM R1–1C (4 h and 16 h), 100 nM R1–3C (4 h and 16 h), 100 nM R1–5C (2 h, 4 h, 8 h and 16 h), 100 nM RMC-4550 (16 h) and 1 μM pomalidomide (5 h). The heatmap colors are scaled with red indicating a decrease in protein abundance (−2 log2 FC) and blue indicating an increase (2 log2 FC) in protein abundance.

Article Snippet: Antibodies used in this study were obtained commercially from the following sources: SHP2 (Bethyl, #A301–544A), Phospho-Thr202/Tyr204-Erk1/2 (CST, #9101), Cereblon (CST, #71810), b-actin (Millipore-Sigma, #A1978), b -tubulin (CST, #2146), GAPDH (CST, #5174).

Techniques: Inhibition

Journal: Acta physiologica (Oxford, England)

Article Title: Short-term fasting of mice elevates circulating fibroblast growth factor 23 (FGF23).

doi: 10.1111/apha.14049

Figure Lengend Snippet: FIGURE 5 Altered NaPiIIa (Slc34a1), 24-hydroxylase (Cyp24a1) expression and αKlotho formation in fasted mice. Arithmetic means ± SEM (n = 5) of relative Slc34a1 (A), Cyp24a1 (C), 1α-hydroxylase (Cyp27b1) (D), and αKlotho (Kl) (E) mRNA abundance normalized to hypoxanthine guanine phosphoribosyl transferase (Hprt) expression in kidneys from fed mice and mice fasted for 16 h. Representative Western Blots and arithmetic means ± SEM of renal NaPiIIa (⁓69 kDa, isoform X1: ⁓59 kDa, isoform X2: ⁓48 kDa; B: n = 7) and Klotho (⁓130 to 135 kDa; F: n = 5) protein abundance relative to loading control β-tubulin (⁓55 kDa) in fed and fasted mice. For all panels, n denotes the number of specimens included. There were no dropouts in any analysis. *p < 0.05, †p < 0.01, ‡p < 0.001 indicate significant differences compared to fed mice. ns, non-significant. (A: Mann–Whitney U test, B–F: unpaired t-test).

Article Snippet: Proteins from UMR106 cells, NRVM, kidney, heart, thymus, bone marrow, pancreas (each 30 μg), or bone (10 μg) were subjected to standard Western Blot procedure using 10% or 12% SDS- PAGE gels or 4%– 20% precast SDS- PAGE gels (for FGF23 detection in NRVM), respectively, and the following primary antibodies: anti- NFκB- p65 (#8242, Cell Signaling Technology), anti- phospho- NFκB- p65 (#3033, Cell Signaling Technology), anti- FGF23 (MAB26291, R&D Systems), anti- FGF23 (#6320, Immutopics), anti- NaPiIIa (NBP2- 13328, Novus Biologicals, Bio- Techne), anti- Klotho (AF1819, R&D Systems), anti- β- actin ((#3700 (for lysates from UMR106 cells) and #8457 (for mouse tissue lysates), Cell Signaling Technology)), anti- GAPDH (#2118, Cell Signaling Technology), or anti- β- tubulin (#2146, Cell Signaling Technology).

Techniques: Expressing, Western Blot, Quantitative Proteomics, Control, MANN-WHITNEY

Journal: Oncotarget

Article Title: An increase in galectin-3 causes cellular unresponsiveness to IFN-γ-induced signal transduction and growth inhibition in gastric cancer cells

doi: 10.18632/oncotarget.7750

Figure Lengend Snippet: A PIP3 MASS ELISA Kit assay and Western blot analysis showing PI3K activity A. and expressions of the indicated proteins B. in AGS cells (IFN-γ insensitive) transfected with shRNA targeting luciferase ( shLuc ) and shRNA targeting galectin-3 ( shGal3 ; clones 1-4) and MKN45 cells (IFN-γ sensitive) transfected with pcDNA3- and pcDNA3-Gal3. For kinase activity, the data are presented as the mean ± SD from three independent experiments. ns, not significant. C. Representative Western blot of the indicated proteins in pcDNA3-, pcDNA3-AKT-, GFP-, GFP-tagged GSK-3β R96A -, pBABE-, pBABE-SHP2 wild-type-, and pBABE-SHP2 D61A -tranfected MKN45 cells (IFN-γ sensitive). For Western blotting, β-actin was used as an internal control. pAKT T, phospho-AKT at Thr308; pAKT S, phospho-AKT at Ser473. A representative dataset from triplicate experiments is shown. D. In cells transfected with or without treatment with IFN-γ or the SHP2 inhibitor, NSC87877, for 6 h, the luciferase reporter assay showed the ratio of IRF1 to control Renilla. The data are presented as the mean ± SD from three independent experiments. ** p < 0.01 and *** p < 0.001 compared to the untreated sample; ## p < 0.01 and ### p < 0.001 compared to the relative control. †† p < 0.01 and ††† p < 0.001 compared to IFN-γ.

Article Snippet: The plasmid expressing GFP-PTEN (ID NM_000314; Plasmid 13039) and its control pcDNA3-GFP (Plasmid 13031); pcDNA3 flag HA AKT1 (Plasmid 9021) and its control pcDNA3 flag HA (Plasmid 1436); and pBABE-puro SHP2 (Plasmid 8329) and pBABE-puro SHP2 D61A (Plasmid 8330) and their control vector pBABE-puro (Plasmid 1764) were purchased from Addgene (Cambridge, MA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Activity Assay, Transfection, shRNA, Luciferase, Clone Assay, Control, Reporter Assay

Journal: Oncotarget

Article Title: An increase in galectin-3 causes cellular unresponsiveness to IFN-γ-induced signal transduction and growth inhibition in gastric cancer cells

doi: 10.18632/oncotarget.7750

Figure Lengend Snippet: A proliferation assay A. and propidium iodide (PI) staining-based flow cytometric analysis B. showing cell growth and apoptosis, respectively, in IFN-γ-treated AGS cells transfected with shRNA targeting luciferase ( shLuc ) and shRNA targeting galectin-3 ( shGal3 ). The data (mean ± SD from three independent experiments) are shown as multiples of change compared to the normalized value of the control sample. ** p < 0.01 and *** p < 0.001. ns, not significant. C. A hypothetical model for aberrant galectin-3 involved in facilitating AKT/GSK-3β/SHP2 signaling to inhibit IFN-γ STAT1/IRF1 signaling and IFN-γ-induced cell growth inhibition and apoptosis.

Article Snippet: The plasmid expressing GFP-PTEN (ID NM_000314; Plasmid 13039) and its control pcDNA3-GFP (Plasmid 13031); pcDNA3 flag HA AKT1 (Plasmid 9021) and its control pcDNA3 flag HA (Plasmid 1436); and pBABE-puro SHP2 (Plasmid 8329) and pBABE-puro SHP2 D61A (Plasmid 8330) and their control vector pBABE-puro (Plasmid 1764) were purchased from Addgene (Cambridge, MA).

Techniques: Proliferation Assay, Staining, Transfection, shRNA, Luciferase, Control, Inhibition

Journal: EMBO Molecular Medicine

Article Title: SFX-01 is therapeutic against myeloproliferative disorders caused by activating mutations in Shp2

doi: 10.1038/s44321-025-00267-7

Figure Lengend Snippet: ( A ) Recombinant human Shp2 activity after 30-min incubation with SFX-01. Data represent mean activity ( ± SEM; n = 3 biological replicates) and were fitted to a one-phase exponential decay curve (gray line; r 2 = 0.987). ( B ) Shp2 activity after incubation with SFX-01 for the indicated times and concentrations. Data shown are mean activity ( ± SEM; n = 3 biological replicates). ( C ) Shp2 activity after 30-minute incubation with or without bisphosphorylated IRS1 and SFX-01. Bar represents mean activity (± SEM; n = 4 biological replicates) and P values calculated by two-way ANOVA with Sîdak post hoc test. ( D ) Representative immunoblots showing SFN-modification of recombinant Shp2 (1.6 nM) following incubation with 1.75 or 0.109 µM SFX-01 for 30 min. ( E ) Precursor isotopic envelop spectrum of 0.1 µM recombinant human Shp2 protein incubated with equimolar SFN for 6 h at 37 °C corresponding to a dithiolethione modification adducted between Cys 333 and Cys 367 . ( F ) Schematic representing the proposed mechanism of Shp2-dithiolethione formation by SFN. The isothiocyanate group reacts with a cysteine residue to form a dithiocarbamate intermediate, which further reacts with a second cysteine residue to yield the dithiolethione modification. ( G ) Immunoblot of immunoprecipitated WT or active site mutant Shp2 and SFN-modification from HEK cells treated with SFX-01. “E” represents non-transfected cells, and 0 h represents untreated cells. The graph represents densitometric analysis of Shp2-SFN adduct formation in WT or mutant Shp2 exposed to SFX-01 for 2 or 4 h. Bars represent mean values (± SEM; n = 3 biological replicates) and P values calculated by two-way ANOVA with Sîdak post hoc test. .

Article Snippet: C459S Shp2 , Addgene , #8382.

Techniques: Recombinant, Activity Assay, Incubation, Western Blot, Modification, Residue, Immunoprecipitation, Mutagenesis, Transfection

Journal: EMBO Molecular Medicine

Article Title: SFX-01 is therapeutic against myeloproliferative disorders caused by activating mutations in Shp2

doi: 10.1038/s44321-025-00267-7

Figure Lengend Snippet: Shp2 phosphatase activity in ( A ) cardiac tissue ( n = 8 mice) and ( B ) spleen of WT ( n = 4 mice) or Ptpn11 D61G (−/+) mice ( n = 5 mice) that received SFX-01 in the drinking water for 10 days. ( A , B ) Bars represent mean activity ( ± SEM) and P values calculated by two-way ANOVA with Sîdak post hoc test. ( C ) Representative immunoblots showing the input and immunoprecipitated Shp2 protein and SFN-labeled Shp2 from cardiac tissue isolated from mice with or without oral SFX-01 administration as in ( A ). ( D ) Representative immunoblot of Shp2 immunoprecipitated from cardiac tissue of WT mice following treatment with SFX-01 for 4 days after incubation with biotin-iodoacetamide. ( E ) Representative far-western immunoblot of Shp2 immunoprecipitated from cardiac tissue of WT mice following treatment with SFX-01 for 4 days. The immunoblot was then exposed to biotin-phenylarsenic acid (PAA). .

Article Snippet: C459S Shp2 , Addgene , #8382.

Techniques: Activity Assay, Western Blot, Immunoprecipitation, Labeling, Isolation, Incubation

Journal: EMBO Molecular Medicine

Article Title: SFX-01 is therapeutic against myeloproliferative disorders caused by activating mutations in Shp2

doi: 10.1038/s44321-025-00267-7

Figure Lengend Snippet: ( A ) Changes in leukocyte cell populations in blood isolated from individual WT or Ptpn11 D61G (−/+) mice before and after 10 weeks of SFX-01 in drinking water. P values were calculated by paired t tests. For WT mice: n = 14 before and after vehicle treatment, n = 14 before SFX-01 treatment, and n = 15 after SFX-01 treatment. For Ptpn11 D61G (−/+) mice: n = 14 before vehicle treatment, n = 16 after vehicle treatment, n = 11 before SFX-01 treatment, and n = 14 after SFX-01 treatment. ( B ) Blood leukocyte cell population after 10 weeks SFX-01 treatment. ( left ) Bars represent mean leukocyte population ( ± SEM) and P values calculated by two-way ANOVA with Sîdak post hoc test. For WT mice: n = 14 vehicle treatment, and n = 15 SFX-01 treatment. For Ptpn11 D61G (−/+) mice: n = 16 vehicle treatment, and n = 14 SFX-01 treatment. ( right ) Representative microphotographs of Wright-Giemsa-stained blood isolated from mice after 10 weeks SFX-01 treatment or controls. Scale bars show 200 µm. ( C ) Violin plots of blood myeloid-derived (CD11b + /Ly6G + and CD11b + /Ly6C + ) cell populations from WT or Ptpn11 D61G (−/+) mice after 10 weeks of SFX-01 in drinking water. Horizontal lines represent quartiles and P values calculated by two-way ANOVA with Sîdak post hoc test. For WT mice: n = 13 vehicle or SFX-01 treatment. For Ptpn11 D61G (−/+) mice: n = 9 vehicle treatment, and n = 13 SFX-01 treatment. ( D ) Changes in spleen size from individual WT or Ptpn11 D61G (−/+) mice before and after 10 weeks of SFX-01 in drinking water. P values were calculated by paired t tests. For WT mice: n = 14 before and after vehicle treatment ( P = 0.0102), n = 14 before and after SFX-01 treatment ( P = 0.0890). For Ptpn11 D61G (−/+) mice: n = 13 before and after vehicle treatment ( P = 0.0253), n = 15 before and after SFX-01 treatment ( P = 0.1694) for each group. ( E ) Mean changes in spleen size in WT or Ptpn11 D61G (−/+) mice over 10 weeks of SFX-01 treatment compared to before treatment. Bars represent mean spleen size ( ± SEM) and P values calculated by two-way ANOVA with Sîdak post hoc test. For WT mice: n = 14 vehicle or SFX-01 treatment. For Ptpn11 D61G (−/+) mice: n = 13 vehicle treatment, and n = 15 SFX-01 treatment. ( F ) Violin plots of spleen myeloid-derived (CD11b + /Ly6G + and CD11b + /Ly6C + ) cell populations from WT or Ptpn11 D61G (−/+) mice after 10 weeks of SFX-01 in drinking water. Horizontal lines represent quartiles and P values calculated by two-way ANOVA with Sîdak post hoc test. For WT mice: n = 14 vehicle treatment, and n = 15 SFX-01 treatment ( P = 0.6138). For Ptpn11 D61G (−/+) mice: n = 16 vehicle treatment, and n = 12 SFX-01 treatment ( P = 0.0251). .

Article Snippet: C459S Shp2 , Addgene , #8382.

Techniques: Isolation, Staining, Derivative Assay

Journal: EMBO Molecular Medicine

Article Title: SFX-01 is therapeutic against myeloproliferative disorders caused by activating mutations in Shp2

doi: 10.1038/s44321-025-00267-7

Figure Lengend Snippet: ( A ) Representative immunoblot of relative STAT1 phosphorylation and cyclin D1 expression in leukocytes isolated from WT or Ptpn11 D61G (−/+) mice after 10-week SFX-01 treatment. Graphs represent densitometric analyses of relative STAT1 (Ser 727) phosphorylation and cyclin D1 expression. Bars represent mean expression level ( ± SEM) and P values calculated by two-way ANOVA with Sîdak post hoc test. n = 5 mice for STAT1 (Ser 727) phosphorylation and n = 3 mice biological replicates for cyclin D1 expression. ( B ) Representative immunoblots of relative STAT1 phosphorylation and cyclin D1 expression in a Shp2-activating mutant expressing human GDM-1 cell line treated with SFX-01 or vehicle. Graph bars represent mean expression levels ( ± SEM; n = 4 biological replicates) and P values calculated by unpaired t test. ( C ) Cell cycle analysis of GDM-1 cells cultured with SFX-01. Bars represent mean percentage ( ± SEM), and significance compared to controls was calculated by one-way ANOVA with Dunnett’s test. n = 4 for vehicle or 250 µM SFX-01 treatment, n = 3 for 1, 5, 25, or 100 µM SFX-01 treatment, biological replicates. ( D ) Colony-forming ability of GDM-1 cells treated with SFX-01. Bars represent mean normalized values (± SEM), and because of the low sample size and not assuming a normal Gaussian distribution, significance was calculated using a Kruskal–Wallis test with an uncorrected Dunn’s test for comparison to the control group. n = 2 for vehicle treatment, n = 3 for 0.1 µM SFX-01 treatment ( P = 0.3957), n = 3 for 1 µM SFX-01 treatment ( P = 0.2167), n = 5 for 10 µM SFX-01 ( P = 0.0245) and n = 3 for 100 µM SFX-01 ( P = 0.0018) treatment, biological replicates. ( E ) Cell cycle analysis and ( F ) colony-forming ability of control (CB) or JMML patient-derived HSCs cultured with or without SFX-01. Clonogenicity of HSC was normalized to control cells, and bars represent mean ( ± SEM) and P values calculated by two-way ANOVA with Sîdak post hoc test. For ( E ): CB: n = 3 for vehicle, 5, or 25 µM SFX-01 treatment, and n = 2 for 100 µM SFX-01 treatment. JMML: n = 7 for vehicle or 5 µM SFX-01 treatment, and n = 8 for 25 or 100 µM SFX-01 treatment, biological replicates. For ( F ): CB: n = 14 for vehicle treatment, n = 4 for 10 µM SFX-01 treatment, n = 17 for 50 µM SFX-01 treatment, and n = 19 for 100 µM SFX-01 treatment. JMML: n = 6 for vehicle or 10 µM SFX-01 treatment, n = 11 for 50 µM SFX-01 treatment, and n = 12 for 100 µM SFX-01 treatment, biological replicates. ( G ) A heatmap displaying the significantly altered Shp2 substrates in CD11b+ myeloid bone marrow cells isolated from WT or Ptpn11 D61G (−/+) mice, under conditions with or without SFX-01 treatment based on data from the phosphoproteomics analysis ( n = 3 biological replicates). .

Article Snippet: C459S Shp2 , Addgene , #8382.

Techniques: Western Blot, Phospho-proteomics, Expressing, Isolation, Mutagenesis, Cell Cycle Assay, Cell Culture, Comparison, Control, Derivative Assay

Journal: EMBO Molecular Medicine

Article Title: SFX-01 is therapeutic against myeloproliferative disorders caused by activating mutations in Shp2

doi: 10.1038/s44321-025-00267-7

Figure Lengend Snippet: CD11b+ bone marrow cells isolated from WT and Ptpn11 D61G (−/+) mice treated with SFX-01, SHP099 or both together. SFX-01 or SHP099 attenuated cell proliferation and this was not accentuated when these interventions were combined. Data are presented as means ( ± SEM; n = 5–6) with P values calculated by two-way ANOVA with Tukey’s multiple comparison test. * P < 0.00001 versus untreated WT or # P < 0.00001 versus untreated Ptpn11 D61G (−/+) .

Article Snippet: C459S Shp2 , Addgene , #8382.

Techniques: Isolation, Comparison

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