Journal: JCI Insight

Article Title: Phase separation of SHP2 E76K promotes malignant transformation of mesenchymal stem cells by activating mitochondrial complexes

doi: 10.1172/jci.insight.170340

Figure Lengend Snippet: ( A and B ) immunofluorescence (IF) images ( A ) and statistical analysis ( B ) of Ki67 staining of WT and SHP2 E76K MSCs ( n = 5 per group). Scale bar, 200 μm. Data are represented as the mean ± SD. ** P < 0.01 (2-tailed unpaired t test). ( C and D ) Microscopy image ( C ) and statistical analysis ( D ) of soft agar colony formation capacity of WT and SHP2 E76K MSCs ( n = 5 per group). Scale bar, 200 μm. Data are represented as the mean ± SD. **** P < 0.01 (2-tailed unpaired t test). ( E ) Xenograft tumor transplantation of WT and SHP2 E76K MSCs in nude mice ( n = 5 per group). ( F ) Statistical analysis of the tumor incidence of WT and SHP2 E76K MSCs in nude mice ( n = 5). ( G ) HE staining and IF staining images of the indicated antibodies in SHP2 E76K -induced sarcoma from nude mice. Scale bar, 200 μm. ( H ) Xenograft tumor transplantation of WT and SHP2 E76K MSCs in immunocompetent (C57BL/6) mice. ( I ) Statistical analysis of the tumor incidence of WT and SHP2 E76K MSCs in C57BL/6 mice ( n = 4 per group). ( J ) HE staining and IF staining of the indicated antibodies in SHP2 E76K -induced sarcoma from C57BL/6 mice. Scale bar, 200 μm. ( K ) Representative IF images of WT and SHP2 E76K human umbilical cord MSCs in the field of 200×. ( L and M ) Representative tumor images ( L ) and statistical analysis ( M ) of tumor incidence in nude mice with subcutaneous inoculation of WT and SHP2 E76K human MSCs ( n = 5 per group). ( N ) Representative HE and immunohistochemical images of tumor developed by SHP2 E76K human umbilical cord MSCs. Scale bar, 500 μm.

Article Snippet: MSCs expressing SHP2 WT and SHP2 E76K were harvested to determine the MMP using a JC-1 assay kit (MedChemExpress, HY-K0601).

Techniques: Immunofluorescence, Staining, Microscopy, Transplantation Assay, Immunohistochemical staining

Journal: JCI Insight

Article Title: Phase separation of SHP2 E76K promotes malignant transformation of mesenchymal stem cells by activating mitochondrial complexes

doi: 10.1172/jci.insight.170340

Figure Lengend Snippet: ( A and B ) Representative images ( A ) and statistical analysis ( B ) of microspheres in WT and SHP2 E76K MSCs ( n = 5 per group). Data are represented as the mean ± SD. ** P < 0.01 (2-tailed unpaired t test). ( C ) Schematic outline of the study design, depicting the workflow of serial transplantation of SHP2 E76K MSCs. ( D ) Statistical analysis of tumor weight following serial transplantation of SHP2 E76K MSCs ( n = 4 per group). Data are represented as the mean ± SD. ** P < 0.01, *** P < 0.001 (2-tailed unpaired t test). ( E and F ) Representative flow cytometry images ( E ) and statistical analysis ( F ) of CD184-positive cells in WT and SHP2 E76K MSCs ( n = 5 per group). Data are represented as the mean ± SD. *** P < 0.001 (2-tailed unpaired t test). ( G and H ) Representative flow cytometry images ( G ) and statistical analysis ( H ) of CD271-positive cells in WT and SHP2 E76K MSCs ( n = 5 per group). Data are represented as the mean ± SD. **** P < 0.0001 (2-tailed unpaired t test). ( I and J ) Representative flow cytometry images ( I ) and statistical analysis ( J ) of CD344-positive cells in WT and SHP2 E76K MSCs ( n = 5 per group). Data are represented as the mean ± SD. **** P < 0.0001 (2-tailed unpaired t test). ( K and L ) Representative flow cytometry images ( K ) and statistical analysis ( L ) of CD133-positive cells in WT and SHP2 E76K MSCs ( n = 5 per group). Data are represented as the mean ± SD. ** P < 0.01 (2-tailed unpaired t test). ( M ) Gating strategy for CD184-positive and CD184-negative SHP2 E76K MSCs’ isolation using FACS. ( N and O ) Representative tumor images ( N ) and statistical analysis of tumor ( O ) generated by CD184-positive and CD184-negative SHP2 E76K MSCs ( n = 3 or 4 per group). Data are represented as the mean ± SD. ** P < 0.01 (2-tailed unpaired t test).

Article Snippet: MSCs expressing SHP2 WT and SHP2 E76K were harvested to determine the MMP using a JC-1 assay kit (MedChemExpress, HY-K0601).

Techniques: Transplantation Assay, Flow Cytometry, Isolation, Generated

Journal: JCI Insight

Article Title: Phase separation of SHP2 E76K promotes malignant transformation of mesenchymal stem cells by activating mitochondrial complexes

doi: 10.1172/jci.insight.170340

Figure Lengend Snippet: ( A ) Volcano plot analysis of WT and SHP2 E76K MSCs ( n = 4). ( B ) KEGG pathway analysis of differentially expressed proteins (DEPs) of WT and SHP2 E76K MSCs. ( C ) Statistical analysis of ATP production in WT and SHP2 E76K MSCs ( n = 6–7 per group). Data are represented as the mean ± SD. **** P < 0.0001 (2-tailed unpaired t test). ( D ) Representative image of OCR analysis in WT and SHP2 E76K MSCs. ( E ) Representative image of ECAR analysis in WT and SHP2 E76K MSCs. ( F and G ) Statistical analysis of mitochondrial respiration of WT and SHP2 E76K MSCs ( n = 5 per group) at basal ( F ) and maximum ( G ) levels. Data are represented as the mean ± SD. **** P < 0.0001 (2-tailed unpaired t test). ( H and I ) Statistical analysis of the glycolytic function of WT and SHP2 E76K MSCs ( n = 5 per group) at basal ( H ) and maximum ( I ) levels. Data are represented as the mean ± SD. **** P < 0.0001 (2-tailed unpaired t test). ( J and K ) Statistical analysis of glucose consumption ( J , n = 3 per group) and glutamate content ( K , n = 7 or 8 per group) in WT and SHP2 E76K MSCs. Data are represented as the mean ± SD. ** P < 0.01, **** P < 0.0001 (2-tailed unpaired t test). ( L ) Statistical analysis of total enrichment of glutamine, glutamate, malate, fumarate, and citrate in WT and SHP2 E76K MSCs ( n = 3 or 4 per group). Data are represented as the mean ± SD. ** P < 0.01, **** P < 0.0001 (2-tailed unpaired t test). ( M ) Statistical analysis of the enrichment of glutamine (M5), glutamate (M5), malate (M4), fumarate (M4), and citrate (M4) in WT and SHP2 E76K MSCs ( n = 3 or 4 per group). Data are represented as the mean ± SD. * P < 0.05, **** P < 0.0001 (2-tailed unpaired t test).

Article Snippet: MSCs expressing SHP2 WT and SHP2 E76K were harvested to determine the MMP using a JC-1 assay kit (MedChemExpress, HY-K0601).

Techniques:

Journal: JCI Insight

Article Title: Phase separation of SHP2 E76K promotes malignant transformation of mesenchymal stem cells by activating mitochondrial complexes

doi: 10.1172/jci.insight.170340

Figure Lengend Snippet: ( A ) Statistical analysis of the activity of complexes I, II, III, and IV in WT and SHP2 E76K MSCs ( n = 3 to 7 per group). Data are represented as the mean ± SD. * P < 0.05 (2-tailed unpaired t test). ( B and C ) Representative images ( B ) and statistical analysis ( C ) of MMP in WT and SHP2 E76K MSCs ( n = 7 per group). Data are represented as the mean ± SD. **** P < 0.0001 (2-tailed unpaired t test). ( D and E ) Representative images ( D ) and statistical analysis ( E ) of ROS production in WT and SHP2 E76K MSCs ( n = 3 per group). Data are represented as the mean ± SD. ** P < 0.01 (2-tailed unpaired t test). ( F ) IF staining images of MitoSOX Red in WT and SHP2 E76K MSCs. ( G ) Statistical analysis of the relative level of mitochondrial ROS in WT and SHP2 E76K MSCs ( n = 6 per group) via flow cytometry. Data are represented as the mean ± SD. ** P < 0.01 (2-tailed unpaired t test).

Article Snippet: MSCs expressing SHP2 WT and SHP2 E76K were harvested to determine the MMP using a JC-1 assay kit (MedChemExpress, HY-K0601).

Techniques: Activity Assay, Staining, Flow Cytometry

Journal: JCI Insight

Article Title: Phase separation of SHP2 E76K promotes malignant transformation of mesenchymal stem cells by activating mitochondrial complexes

doi: 10.1172/jci.insight.170340

Figure Lengend Snippet: ( A ) Schematic diagram depicting the effect of metformin and atovaquone on malignant transformation in SHP2 E76K MSCs. ( B ) Representative image of OCR analysis in SHP2 E76K MSCs treated with metformin and atovaquone. ( C and D ) Statistical analysis of mitochondrial respiration in SHP2 E76K MSCs at basal ( C ) and maximum levels ( D ) following treatment with metformin or atovaquone ( n = 4 per group). Data are represented as the mean ± SD. * P < 0.05, ** P < 0.01, **** P < 0.0001 (1-way ANOVA with multiple-comparison test). ( E – H ) Statistical analysis of glucose consumption ( E ), glutamate ( F ), MMP ( G ), and ROS ( H ) in SHP2 E76K MSCs ( n = 4 or 5 per group) treated with metformin or atovaquone. Data are represented as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (1-way ANOVA with multiple-comparison test). ( I ) Statistical analysis of soft agar colony formation of SHP2 E76K MSCs treated with metformin or atovaquone ( n = 5 per group). Data are represented as the mean ± SD. *** P < 0.001 (1-way ANOVA with multiple-comparison test). ( J ) Statistical analysis of spheroid formation of SHP2 E76K MSCs treated with metformin or atovaquone ( n = 5 per group). Data are represented as the mean ± SD. **** P < 0.0001 (1-way ANOVA with multiple-comparison test). ( K ) Statistical analysis of percentage of CD184 + SHP2 E76K MSCs treated with metformin or atovaquone ( n = 5 per group). Data are represented as the mean ± SD. *** P < 0.001, **** P < 0.0001 (1-way ANOVA with multiple-comparison test). ( L ) Statistical analysis of tumor formation of SHP2 E76K MSCs treated with metformin or atovaquone ( n = 4 per group). Data are represented as the mean ± SD. **** P < 0.0001 (1-way ANOVA with multiple-comparison test).

Article Snippet: MSCs expressing SHP2 WT and SHP2 E76K were harvested to determine the MMP using a JC-1 assay kit (MedChemExpress, HY-K0601).

Techniques: Transformation Assay, Comparison

Journal: JCI Insight

Article Title: Phase separation of SHP2 E76K promotes malignant transformation of mesenchymal stem cells by activating mitochondrial complexes

doi: 10.1172/jci.insight.170340

Figure Lengend Snippet: ( A ) WT and SHP2 E76K MSC lysates were examined by immunoprecipitation and immunoblotting as indicated. ( B and C ) Representative IF staining images ( B ) and statistical analysis ( C ) of SHP2 droplets in WT and SHP2 E76K MSCs ( n = 6 per group). Data are represented as the mean ± SD. *** P < 0.001 (2-tailed unpaired t test). Scale bar, 15 μm. ( D ) Workflow of the establishment of a mouse model harboring a SHP2 triple mutation (SHP2 E76K/R362E/K364E ). ( E and F ) Representative IF staining images ( E ) and statistical analysis ( F ) of SHP2 droplets in SHP2 E76K and SHP2 E76K/R362E/K364E MSCs ( n = 6 per group). Data are represented as the mean ± SD. **** P < 0.0001 (2-tailed unpaired t test). Scale bar, 15 μm. ( G ) Immunoprecipitation of SHP2 in SHP2 E76K and SHP2 E76K/R362E/K364E MSCs ( n = 5 per group) and analyzed using immunoblot analysis with the indicated antibodies. ( H ) Statistical analysis of complex I and complex III activity in SHP2 E76K MSCs with and without an additional R362E/K364E mutation. Data are represented as the mean ± SD. ** P < 0.01, *** P < 0.001 (2-tailed unpaired t test). ( I and J ) Statistical analysis of complex I ( I ) and complex III ( J ) activity in WT and SHP2 E76K HEK293T cells following the R362E/K364E mutation ( n = 3 or 5 per group). Data are represented as the mean ± SD. ** P < 0.01, *** P < 0.001 (2-way ANOVA with multiple-comparison test). ( K – N ) Statistical analysis of soft agar colony formation ( K ), microsphere formation ( L ), percentage of CD184 + cells (M), and sarcoma weights ( N ) in SHP2 E76K and SHP2 E76K/R362E/K364E MSCs. Data are represented as the mean ± SD. ** P < 0.01, **** P < 0.0001 (2-tailed unpaired t test).

Article Snippet: MSCs expressing SHP2 WT and SHP2 E76K were harvested to determine the MMP using a JC-1 assay kit (MedChemExpress, HY-K0601).

Techniques: Immunoprecipitation, Western Blot, Staining, Mutagenesis, Activity Assay, Comparison

Journal: JCI Insight

Article Title: Phase separation of SHP2 E76K promotes malignant transformation of mesenchymal stem cells by activating mitochondrial complexes

doi: 10.1172/jci.insight.170340

Figure Lengend Snippet: ( A ) Schematic diagram depicting the effect of SHP2 inhibitors on mitochondrial complexes I and III and malignant transformation in SHP2 E76K MSCs. ( B and C ) Representative IF images ( B ) and statistical analysis ( C ) of SHP2 droplets in SHP2 E76K MSCs treated with SHP099 and ET070 ( n = 3 per group). Data are represented as the mean ± SD. *** P < 0.001 (1-way ANOVA with multiple-comparison test). ( D ) Immunoprecipitation of SHP2 in WT and SHP2 E76K MSCs with or without SHP099 treatment and analyzed using immunoblot analysis with the indicated antibodies. ( E and F ) Statistical analysis of complex I ( E ) and complex III activity ( F ) in WT and SHP2 E76K MSCs ( n = 4 or 5 per group) treated with SHP099 and ET070. Data are represented as the mean ± SD. ** P < 0.01, *** P < 0.001, **** P < 0.0001 (1-way ANOVA with multiple-comparison test). ( G – I ) Statistical analysis of glucose consumption ( G ), MMP ( H ), and ROS ( I ) in SHP2 E76K MSCs treated with SHP099 or ET070. Data are represented as the mean ± SD. * P < 0.05, *** P < 0.001, **** P < 0.0001 (1-way ANOVA with multiple-comparison test). ( J ) Statistical analysis of tumor spheroids in SHP2 E76K MSCs following SHP099 and ET070 treatment. Data are represented as the mean ± SD. ** P < 0.01, *** P < 0.001 (1-way ANOVA with multiple-comparison test). ( K and L ) Representative images ( K ) and incidence ( L ) of tumors induced by SHP2 E76K MSCs treated with or without SHP2 allosteric inhibitors (i.e., SHP099 or ET070) ( n = 10 per group).

Article Snippet: MSCs expressing SHP2 WT and SHP2 E76K were harvested to determine the MMP using a JC-1 assay kit (MedChemExpress, HY-K0601).

Techniques: Transformation Assay, Comparison, Immunoprecipitation, Western Blot, Activity 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 ) 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: Protein-containing samples in SDS-PAGE sample buffer were subjected to electrophoresis and immunoblotting using the following primary antibodies: SFN (1:1000, in-house), Shp2 (1:1000, Abcam #32083) for recombinant protein or (R&D Systems #AF1894) for immunoprecipitation experiments, GAPDH (1:5000, CST #2118) and an anti-rabbit secondary antibody (1:2500, CST #7074).

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

Journal: Nature cell biology

Article Title: Dynamic regulation of integrin β1 phosphorylation supports invasion of breast cancer cells.

doi: 10.1038/s41556-025-01663-4

Figure Lengend Snippet: Fig. 5 | ITGB1 is a substrate for PTP-PEST and Shp2. a,b, A malachite green assay for free phosphate release after incubation of phosphorylated/non- phosphorylated ITGB1 peptides with recombinant Shp2 (n = 5 independent replicates, each performed in triplicate) (a) or PTP-PEST (n = 4 independent replicates, each performed in triplicate) (b). The significance was assessed using a Kruskal–Wallis test with a Dunn’s correction for multiple comparisons. The data are presented as the mean ± s.e.m. c,d, Schematics of FRET experiments (left) using mRuby2-tagged ITGB1 and Clover-tagged PTPs. Representative FLIM–FRET images (right) and quantification of apparent FRET efficiency of MM231 cells with stable expression of either ITGB1(WT)–mRuby2 or ITGB1(YYFF)–mRuby2

Article Snippet: Each fragment (2,400 pmol per peptide per reaction) was incubated separately for 1 h at 37 °C with recombinant Shp2 (0.05 μg ml−1; R&D Systems, 1894-SH-100) or PTP-PEST (0.05 μg ml−1; SignalChem, P39-21G-10) in phosphatase buffer (HEPES buffer, pH 7.5 (50 mM)/EDTA (0.2 mM)/DTT (5 mM)/Triton X-100 (0.01%)), before incubation with Malachite Green Reagent (100 μl per reaction).

Techniques: Malachite Green Assay, Incubation, Recombinant, Expressing

Journal: Nature Communications

Article Title: The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis

doi: 10.1038/s41467-018-05768-3

Figure Lengend Snippet: Decreased expression of SHP2 in SSc. a The mRNA levels of SHP2 are significantly reduced in SSc skin as compared to healthy skin ( n = 7). b Immunohistochemistry of SHP2 in SSc skin and matched healthy controls. Representative images are shown at 200- and 1000-fold magnification. c Immunofluorescence staining of SHP2 with co-staining for the fibroblast marker P4Hβ, the endothelial cell marker CD31 and the leukocyte marker CD45, and DAPI. SSc fibroblasts demonstrated a reduced staining for SHP2 compared to healthy control. Representative images are shown at 400-fold magnification. Immunofluorescence pictures were processed to generate Voronoi tessellated pictures amenable to computational simulation. Quantification of SHP2 staining intensity ( n = 5) and of SHP2-positive cells ( n = 5). d , e The mRNA ( n = 5) ( d ) and protein level ( n = 4) ( e ) of SHP2 are decreased in cultured SSc fibroblasts. Horizontal scale bar, for all images, 500 μm. All data are presented as median ± s.e.m. The p values are expressed as follows: 0.05 > p > 0.01*; 0.01 > p > 0.001**; p < 0.001***. Significance was determined by Mann–Whitney test. SSc: systemic sclerosis, Healthy: healthy individual, int.: intensity

Article Snippet: A Recombinant Human SHP2 (R&D Systems, Minneapolis, USA) was used to generate a standard curve (0, 1, 2, 4, 6, 8 and 10 ng).

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Staining, Marker, Control, Cell Culture, MANN-WHITNEY

Journal: Nature Communications

Article Title: The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis

doi: 10.1038/s41467-018-05768-3

Figure Lengend Snippet: SHP2 is downregulated in TGFβ signaling. a , b Decreased mRNA ( n = 6) ( a ) and protein ( n = 4) ( b ) levels of SHP2 in healthy fibroblasts stimulated with TGFβ (10 ng/ml) for different time points as measured by RT-PCR and western blot, respectively. c , d Overexpression of TBRI CA (6.67 × 10 7 IFUs every 2 weeks) significantly reduced mRNA ( n = 8) and the protein levels of Shp2 in murine skin as shown by qPCR ( c ) and immunofluorescence staining ( d ) of Shp2 with co-staining for fibroblast marker Vimentin and DAPI ( n ≥ 6 per each group). Representative images are shown at 100–200- and 600-fold magnification. Horizontal scale bar, 500 μm. Immunofluorescence pictures were analyzed by Voronoi tessellation. e , f Treatment with the selective TGFβ receptor type 1 kinase inhibitor SD208 (60 mg/kg/day) reversed the decrease of Shp2 mRNA ( n = 6) ( e ) and protein ( n = 4) ( f ) in bleomycin-challenged mice (50 µg every other day). g , h Treatment with the selective TGFβ receptor type 1 kinase inhibitor SD-208 reversed the decrease of Shp2 mRNA ( n = 6) ( g ) and protein ( h ) in TSK1 mice (2 mg tamoxifen over 5 days) ( n ≥ 6 per each group). i Phosphatase activity assay. Increases in SHP2 activity after TGFβ stimulation (10 ng/ml) ( n = 4) in cultured fibroblasts and upon overexpression of TGFβRI (6.67 × 10 7 IFUs) in murine skin ( n ≥ 4 per each group). Results shown are representative of three independent experiments. All data are presented as median ± s.e.m. The p values are expressed as follows: 0.05 > p > 0.01*; 0.01 > p > 0.001**; p < 0.001***. Significance was determined by Mann–Whitney test. AdLacZ: adenovirus LacZ, TBRI CA : constitutively active TGFβ receptor type I, TSK1: Tight skin, Bleo: bleomycin, Pa/Pa: control for TSK1, fluo.: fluorescence, int.: intensity, Unst.: unstimulated

Article Snippet: A Recombinant Human SHP2 (R&D Systems, Minneapolis, USA) was used to generate a standard curve (0, 1, 2, 4, 6, 8 and 10 ng).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Over Expression, Immunofluorescence, Staining, Marker, Phosphatase Assay, Activity Assay, Cell Culture, MANN-WHITNEY, Control, Fluorescence

Journal: Nature Communications

Article Title: The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis

doi: 10.1038/s41467-018-05768-3

Figure Lengend Snippet: Shp2 regulates TGFβ induced fibroblast activation. a Western blot for efficiency of Cre-mediated (80 IFUs/cell) knockout of Shp2 in murine Shp2 fl/fl fibroblasts ( n ≥ 3 per each group). b Shp2 knockout decreased mRNA levels of Acta2 ( n = 6). c – e Shp2 knockout decreased α-SMA and stress fiber staining. Representative images are shown at 200-fold magnification ( c ). Horizontal scale bar, 500 μm. Quantification of α-SMA staining intensity ( d ) and stress fiber staining intensity ( e ) ( n ≥ 3 different lines). f , g Col1a1 mRNA ( f ) and collagen protein release ( g ) induced by TGFβ (10 ng/ml for 24 h) ( n ≥ 3 different lines). Results shown are representative of three independent experiments. All data are presented as median ± s.e.m. The p values are expressed as follows: 0.05 > p > 0.01* or # ; 0.01 > p > 0.001**; p < 0.001*** or ### ; ns: not significant. Significance was determined by Mann–Whitney test. AdCre: adenovirus Cre, AdLacZ: adenovirus LacZ, int.: intensity

Article Snippet: A Recombinant Human SHP2 (R&D Systems, Minneapolis, USA) was used to generate a standard curve (0, 1, 2, 4, 6, 8 and 10 ng).

Techniques: Activation Assay, Western Blot, Knock-Out, Staining, MANN-WHITNEY

Journal: Nature Communications

Article Title: The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis

doi: 10.1038/s41467-018-05768-3

Figure Lengend Snippet: Fibroblast-specific knockout of Shp2 protects from experimental fibrosis. a TBRI CA -induced fibrosis (6.67 × 10 7 IFUs every 2 weeks). Representative images of Masson trichrome-stained skin shown at 100-fold magnification. Dermal thickness, hydroxyproline content and myofibroblast counts. All groups consisted of ≥9 mice each. b Bleomycin-induced skin (50 µg every other day) fibrosis. Representative images of Masson trichrome-stained skin shown at 100-fold magnification. Dermal thickness, hydroxyproline content and myofibroblast counts. All groups consisted of ≥8 mice each. c TSK1 model (2 mg tamoxifen over 5 days). Representative images of Masson trichrome-stained skin shown at 40-fold magnification. Hypodermal thickness, hydroxyproline content and myofibroblast counts. All groups consisted of ≥8 mice each. Horizontal scale bar in all images, 500 μm. All data are presented as median ± s.e.m. The p values are expressed as follows: 0.05 > p > 0.01*; 0.01 > p > 0.001** or ## ; p < 0.001***; ns: not significant. AdLacZ: adenovirus LacZ, TBRI CA : constitutively active TGFβ receptor type I, TSK1: Tight skin, Bleo: bleomycin, Shp2 Ko: SHP2 fibroblast-specific knockout

Article Snippet: A Recombinant Human SHP2 (R&D Systems, Minneapolis, USA) was used to generate a standard curve (0, 1, 2, 4, 6, 8 and 10 ng).

Techniques: Knock-Out, Staining

Journal: Nature Communications

Article Title: The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis

doi: 10.1038/s41467-018-05768-3

Figure Lengend Snippet: Knockout of Shp2 decreases JAK2/STAT3 signaling. a Knockout of Shp2 in fibroblasts ( Shp2 fl/fl x Col1a2 ; Cre ER) decreases the levels of pJAK2 and pSTAT3 and STAT3 reporter activity in cultured fibroblasts ( n = 3 different lines). Cells were stimulated with TGFβ (10 ng/ml for 6 h). b – d Conditional knockout of Shp2 reduces the levels of pJAK2 and pSTAT3 in TBRI CA (6.67 × 10 7 IFUs every 2 weeks) ( b ) and bleomycin-induced fibrosis (50 µg every other day) ( c ) and in TSK1 mice (2 mg tamoxifen over 5 days) ( d ) ( n ≥ 3). Results shown are representative of three independent experiments. All data are presented as median ± s.e.m. The p values are expressed as follows: 0.05 > p > 0.01* or # ; 0.01 > p > 0.001** or ## ; p < 0.001***; ns: not significant. Significance was determined by Mann–Whitney test. AdLacZ or LacZ: adenovirus LacZ, TBRI CA or TBRI: constitutively active TGFβ receptor type I, TSK1: Tight skin, Bleo: bleomycin, Tam: tamoxifen, Co: Control unstimulated, AdCre: adenovirus Cre

Article Snippet: A Recombinant Human SHP2 (R&D Systems, Minneapolis, USA) was used to generate a standard curve (0, 1, 2, 4, 6, 8 and 10 ng).

Techniques: Knock-Out, Activity Assay, Cell Culture, MANN-WHITNEY, Control

Journal: Nature Communications

Article Title: The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis

doi: 10.1038/s41467-018-05768-3

Figure Lengend Snippet: SHP2 enhances TGFβ-induced fibroblast activation via JAK2/STAT3. a mRNA levels of SHP2 after overexpression in human dermal fibroblasts. mRNA levels of COL1A1 in human fibroblasts transfected with empty vector, SHP2 WT - and SHP2 C459S -expression vectors, with or without TGFβ1 treatment (10 ng/ml for 24 h) ( n ≥ 4). b Western blot analysis and respective quantifications for type I collagen and SHP2 in human fibroblasts transfected with empty vector, SHP2 WT - and SHP2 C459S -expression vectors, with or without TGFβ1 treatment (10 ng/ml for 24 h). Western blot for pJAK2 Y1007/Y1008 , pJAK2 Y570 , total JAK2, pSTAT3 Y705 and total STAT3 with β-actin as loading control (TGFβ 10 ng/ml for 6 h) ( n = 3). Results shown are representative of three independent experiments. c , d Representative images of immunofluorescence stainings for α-SMA and stress fiber staining are shown at 400-fold magnification ( c ) and quantification of α-SMA staining intensity as well as stress fiber staining intensity ( d ) ( n ≥ 4). Horizontal scale bar, 500 μm. All data are presented as median ± s.e.m. The p values are expressed as follows: 0.05 > p > 0.01*; 0.01 > p > 0.001**; p < 0.001***. Significance was determined by Mann–Whitney test. Vector: empty vector, SHP2 WT : plasmid carrying full length of SHP2 wild-type gene, SHP2 C459S : plasmid carrying a phosphatase-dead mutant of SHP2 , unstim.: unstimulated, int.: intensity

Article Snippet: A Recombinant Human SHP2 (R&D Systems, Minneapolis, USA) was used to generate a standard curve (0, 1, 2, 4, 6, 8 and 10 ng).

Techniques: Activation Assay, Over Expression, Transfection, Plasmid Preparation, Expressing, Western Blot, Control, Immunofluorescence, Staining, MANN-WHITNEY, Mutagenesis

Journal: Nature Communications

Article Title: The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis

doi: 10.1038/s41467-018-05768-3

Figure Lengend Snippet: Overexpression of JAK2 ∆Y570F prevents the inhibitory effects of SHP2 inhibitors on TGFβ-induced fibroblast activation. a mRNA levels of COL1A1 and COL1A2 (TGFβ 10 ng/ml for 24 h) ( n ≥ 5). b Release of collagen protein (TGFβ 10 ng/ml for 24 h) ( n ≥ 6). c Representative images of immunofluorescence stainings for α-SMA and stress fiber at 400-fold magnification and respective quantifications (TGFβ 10 ng/ml for 24 h) ( n ≥ 6). Horizontal scale bar, 500 μm. d STAT3 reporter Assay upon JAK2 WT and Y570F mutant overexpression. Cells were treated with TGFβ (10 ng/ml for 6 h) and NSC-87877 (100 µM) ( n ≥ 4). e Co-immunoprecipitation and respective quantifications of endogenous JAK2 with endogenous SHP2 in human fibroblasts stimulated with TGFβ (10 ng/ml for 30′) ( n = 3). Results shown are representative of ≥ 3 independent experiments. All data are presented as median ± s.e.m. The p values are expressed as follows: 0.05 > p > 0.01*; 0.01 > p > 0.001**; p < 0.001***; ns: not significant. Significance was determined by Mann–Whitney test. JAK2 WT : JAK2 Wild type, JAK2 ΔY570F : JAK2 mutant resistant to phosphorylation at Y570, NSC-87877: SHP1/SHP2 inhibitor, Co: control unstimulated, int.: intensity, IP: immunprecipitation

Article Snippet: A Recombinant Human SHP2 (R&D Systems, Minneapolis, USA) was used to generate a standard curve (0, 1, 2, 4, 6, 8 and 10 ng).

Techniques: Over Expression, Activation Assay, Immunofluorescence, Reporter Assay, Mutagenesis, Immunoprecipitation, MANN-WHITNEY, Phospho-proteomics, Control

Journal: Nature Communications

Article Title: The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis

doi: 10.1038/s41467-018-05768-3

Figure Lengend Snippet: Inhibition of SHP2 limits JAK2/STAT3 signaling and fibroblast activation. a Changes in the mRNA levels of COL1A1 and of collagen protein in human fibroblasts incubated with increasing doses of NSC-87877 (10 µM, 30 µM and 100 µM). Fibroblasts were treated with TGFβ (10 ng/ml) for 24 h. b ACTA2 mRNA. ( n ≥ 4) c Representative images of immunofluorescence stainings for α-SMA and stress fiber staining are shown at 400-fold magnification and quantification of α-SMA staining intensity as well as stress fiber staining intensity ( n ≥ 15) (TGFβ 10 ng/ml for 24 h). Horizontal scale bar, 500 μm. d Representative western blots for pJAK2 Y1007/Y1008 , pJAK2 Y570 , total JAK2, pSTAT3 Y705 and total STAT3 with β-actin as loading control and quantification of the results (TGFβ 10 ng/ml for 6 h) ( n ≥ 2). e Changes in STAT3 reporter activity ( n ≥ 6) (TGFβ 10 ng/ml for 6 h). Results shown are representative of three independent experiments All data are presented as median ± s.e.m. The p values are expressed as follows: 0.05 > p > 0.01*; 0.01 > p > 0.001**; p < 0.001***; ns: not significant. Significance was determined by Mann–Whitney test. NSC-87877: SHP1/SHP2 inhibitor, Co: control unstimulated, int.: intensity

Article Snippet: A Recombinant Human SHP2 (R&D Systems, Minneapolis, USA) was used to generate a standard curve (0, 1, 2, 4, 6, 8 and 10 ng).

Techniques: Inhibition, Activation Assay, Incubation, Immunofluorescence, Staining, Western Blot, Control, Activity Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis

doi: 10.1038/s41467-018-05768-3

Figure Lengend Snippet: Treatment with NSC-87877 ameliorates experimental fibrosis. The SHP1/SHP2 inhibitor NSC-87877 was applied at doses of 5 mg/kg q.d. a Bleomycin-induced skin (50 µg every other day) fibrosis: representative images of Masson trichrome-stained skin shown at 100-fold magnification. Dermal thickness, hydroxyproline content and myofibroblast counts. b TBRI CA -induced (6.67 × 10 7 IFUs every 2 weeks) skin fibrosis: representative images of Masson trichrome-stained skin shown at 100-fold magnification. Dermal thickness, hydroxyproline content and myofibroblast counts. c Bleomycin-induced lung fibrosis (50 µg single doses): representative images of Sirius red-stained lung shown at 100-fold magnification. Quantification of Sirius red-positive area (fibrotic area), hydroxyproline content and myofibroblast counts. All groups in all models consisted of ≥5 mice each. Horizontal scale bar in all images, 500 μm. All data are presented as median ± s.e.m. The p values are expressed as follows: 0.05 > p > 0.01*; 0.01 > p > 0.001**; p < 0.001***. Significance was determined by Mann–Whitney test; Bleo: bleomycin, TBRI CA : constitutively active TGFβ receptor type I, AdLacZ: adenovirus LacZ

Article Snippet: A Recombinant Human SHP2 (R&D Systems, Minneapolis, USA) was used to generate a standard curve (0, 1, 2, 4, 6, 8 and 10 ng).

Techniques: Staining, MANN-WHITNEY

Journal: Nature Communications

Article Title: The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis

doi: 10.1038/s41467-018-05768-3

Figure Lengend Snippet: Selective inhibition of Shp2 ameliorates experimental fibrosis. The following doses of SHP2 inhibitors were applied: PHPS1 (5 mg/kg q.d.), SHP099 (75 mg/kg q.d.) and 11-a1 (7.5 mg/kg q.d.). a Bleomycin-induced pulmonary fibrosis (50 µg single doses) : representative images of Masson trichrome-stained skin shown at 100-fold magnification; fibrotic area, hydroxyproline content and myofibroblast counts. b TBRI CA -induced dermal fibrosis (6.67 × 10 7 IFUs every 2 weeks): representative images of Masson trichrome-stained skin shown at 100-fold magnification; Dermal thickness, myofibroblast counts and hydroxyproline content. All groups in both models consisted of ≥4 mice each. Horizontal scale bar for all images, 500 μm All data are presented as median ± s.e.m. The p values are expressed as follows: 0.05 > p > 0.01*; 0.01 > p > 0.001**; p < 0.001***. Significance was determined by Mann–Whitney test. Bleo: bleomycin, AdLacZ: adenovirus LacZ, TBRI CA : constitutively active TGFβ receptor type I

Article Snippet: A Recombinant Human SHP2 (R&D Systems, Minneapolis, USA) was used to generate a standard curve (0, 1, 2, 4, 6, 8 and 10 ng).

Techniques: Inhibition, Staining, MANN-WHITNEY

Journal: Journal of Gynecologic Oncology

Article Title: TET3-mediated DNA demethylation modification activates SHP2 expression to promote endometrial cancer progression through the EGFR/ERK pathway

doi: 10.3802/jgo.2024.35.e64

Figure Lengend Snippet: ANOVA, analysis of variance; EC, endometrial cancer; EEC, endometrial epithelial cell; NC, negative control; OD, optical density; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; SD, standard deviation; SHP2, src homology phosphotyrosin phosphatase 2. * p<0.05.

Article Snippet: We purchased lentiviral-encapsulated shRNAs targeting SHP2(sh-SHP2 #1, sh-SHP2 #2, sh-SHP2 #3) or TET3 (sh-TET3 #1, sh-TET3 #2, sh-TET3 #3) from OriGene Technologies (Beijing, China).

Techniques: Negative Control, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Journal of Gynecologic Oncology

Article Title: TET3-mediated DNA demethylation modification activates SHP2 expression to promote endometrial cancer progression through the EGFR/ERK pathway

doi: 10.3802/jgo.2024.35.e64

Figure Lengend Snippet: EC, endometrial cancer; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; IC50, median inhibition concentration; MPA, medroxyprogesterone acetate; NC, negative control; SD, standard deviation; SHP2, src homology phosphotyrosin phosphatase 2. *,† p<0.05.

Article Snippet: We purchased lentiviral-encapsulated shRNAs targeting SHP2(sh-SHP2 #1, sh-SHP2 #2, sh-SHP2 #3) or TET3 (sh-TET3 #1, sh-TET3 #2, sh-TET3 #3) from OriGene Technologies (Beijing, China).

Techniques: Inhibition, Concentration Assay, Negative Control, Standard Deviation

Journal: Journal of Gynecologic Oncology

Article Title: TET3-mediated DNA demethylation modification activates SHP2 expression to promote endometrial cancer progression through the EGFR/ERK pathway

doi: 10.3802/jgo.2024.35.e64

Figure Lengend Snippet: EC, endometrial cancer; 5hmC, 5-hydroxymethylcytosine; IgG, immunoglobulin G; NC, negative control; NCBI, National Center for Biotechnology Information; qPCR, quantitative polymerase chain reaction; SD, standard deviation; SHP2, src homology phosphotyrosin phosphatase 2; TET, ten-eleven translocation. * p<0.05.

Article Snippet: We purchased lentiviral-encapsulated shRNAs targeting SHP2(sh-SHP2 #1, sh-SHP2 #2, sh-SHP2 #3) or TET3 (sh-TET3 #1, sh-TET3 #2, sh-TET3 #3) from OriGene Technologies (Beijing, China).

Techniques: Negative Control, Real-time Polymerase Chain Reaction, Standard Deviation, Translocation Assay

Journal: Journal of Gynecologic Oncology

Article Title: TET3-mediated DNA demethylation modification activates SHP2 expression to promote endometrial cancer progression through the EGFR/ERK pathway

doi: 10.3802/jgo.2024.35.e64

Figure Lengend Snippet: EC, endometrial cancer; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; IC50, median inhibition concentration; MPA, medroxyprogesterone acetate; NC, negative control; SD, standard deviation; SHP2, src homology phosphotyrosin phosphatase 2; TET3, ten-eleven translocation 3. *,† p<0.05.

Article Snippet: We purchased lentiviral-encapsulated shRNAs targeting SHP2(sh-SHP2 #1, sh-SHP2 #2, sh-SHP2 #3) or TET3 (sh-TET3 #1, sh-TET3 #2, sh-TET3 #3) from OriGene Technologies (Beijing, China).

Techniques: Inhibition, Concentration Assay, Negative Control, Standard Deviation, Translocation Assay

Journal: Journal of Gynecologic Oncology

Article Title: TET3-mediated DNA demethylation modification activates SHP2 expression to promote endometrial cancer progression through the EGFR/ERK pathway

doi: 10.3802/jgo.2024.35.e64

Figure Lengend Snippet: EC, endometrial cancer; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; MPA, medroxyprogesterone acetate; NC, negative control; SHP2, src homology phosphotyrosin phosphatase 2; TET3, ten-eleven translocation 3. *,† p<0.05.

Article Snippet: We purchased lentiviral-encapsulated shRNAs targeting SHP2(sh-SHP2 #1, sh-SHP2 #2, sh-SHP2 #3) or TET3 (sh-TET3 #1, sh-TET3 #2, sh-TET3 #3) from OriGene Technologies (Beijing, China).

Techniques: Negative Control, Translocation Assay

Journal: Scientific reports

Article Title: Gene signatures and genotype-phenotype correlations of sensorineural hearing loss in Noonan syndrome and related RASopathies.

doi: 10.1038/s41598-025-90635-7

Figure Lengend Snippet: Fig. 1. A flow chart depicting Noonan syndrome and related RASopathies (NS-RAS), and percentages and inheritance patterns of each gene are illustrated. (A) A flow chart illustrating the diagnostic process of clinically diagnosed and genetically confirmed NS-RAS patients. (B) The percentages of 10 causative genes for NS-RAS identified in our study cohort are illustrated. The PTPN11 variant accounts for the largest proportion (54.3%). (C) The inheritance patterns for each gene and the number of patients corresponding to each inheritance pattern among individuals with NS-RAS included in our study are delineated. The most frequent inheritance pattern observed was autosomal dominant de novo (79.8%), followed by autosomal dominant (19.1%), and autosomal recessive (1.1%).

Article Snippet: Plasmids, cell culture, and transfection A human PTPN11 cDNA clone (SC112718) was purchased from Origene.

Techniques: Diagnostic Assay, Variant Assay

Journal: Scientific reports

Article Title: Gene signatures and genotype-phenotype correlations of sensorineural hearing loss in Noonan syndrome and related RASopathies.

doi: 10.1038/s41598-025-90635-7

Figure Lengend Snippet: Fig. 2. Clinical profiles of the PTPN11 variant cohort between those with hearing loss (hearing loss group) and without hearing loss (normal hearing group) as well as within the overall cohort were compared. (A) Among the total cohort, 18 (19.1%) exhibited hearing loss, predominantly with PTPN11 (N = 16, 88.9%) and RAF1 (N = 2, 11.1%) variants. (B–K) A comparative analysis of the prevalence of clinical phenotypes (i.e. craniofacial anomaly, skeletal anomaly, cardiac defects, pectus deformity, skin/hair anomaly, multiple lentigines, intellectual disability, cryptorchidism in male, malignancy, and coagulation disorder) between patients with and without hearing loss using an in-house database are illustrated within the PTPN11 cohort.

Article Snippet: Plasmids, cell culture, and transfection A human PTPN11 cDNA clone (SC112718) was purchased from Origene.

Techniques: Variant Assay, Coagulation

Journal: Scientific reports

Article Title: Gene signatures and genotype-phenotype correlations of sensorineural hearing loss in Noonan syndrome and related RASopathies.

doi: 10.1038/s41598-025-90635-7

Figure Lengend Snippet: Fig. 4. Distinct auditory phenotypes in PTPN11 variants. (A) The figure illustrates the distribution of patients with SNHL and normal hearing for all variants of PTPN11 (B) The penetrance and expressivity of SNHL for four variants of PTPN11 (p.Glu139Asp, p.Tyr279Cys, p.Asn308Asp, and p.Gly503Arg).

Article Snippet: Plasmids, cell culture, and transfection A human PTPN11 cDNA clone (SC112718) was purchased from Origene.

Techniques:

Journal: Journal of Alzheimer's disease : JAD

Article Title: Small Molecule Amyloid-β Protein Precursor Processing Modulators Lower Amyloid-β Peptide Levels via cKit Signaling

doi: 10.3233/JAD-180923

Figure Lengend Snippet: Y10 enhances AβPP phosphorylation via cKit. A) IP-WB analysis of the effects of Y10 at 1 μM on AβPP phosphorylation of empty vector or cKit-transfected AβPP stable cells. B) Statistical analysis of (A). Results normalized to AβPP input. C) IP-WB analysis of the effects of Y10 on inhibition of cKit phosphorylation. D) Statistical analysis of (C). Results normalized to cKit input. E) In addition to Y10, a specific inhibitor of cKit, AB1010, also enhances AβPP phosphorylation on Tyr residue. F) Statistical analysis of (E). Results normalized to AβPP input. p-Y, phosphorylated Tyr. G, H) IP-WB analysis of the dose-dependent effects of Y10 and AB1010 on AβPP phosphorylation of cKit-transfected HEK cells stably expressing AβPP. I) Y10 and AB1010 were assayed at 1 μM for 18 h for their effects on Aβ40 level, as measured by ELISA in HEK293 cells stably overexpressing AβPP751. N=17 for Y10 and N=8 for AB1010. Standard error indicated. J) IP-WB analysis of the effects of Y10 at 1 μM on AβPP phosphorylation of control siRNA or cKit siRNA transfected HEK293 cells stably expressing AβPP. K) Statistical analysis of J for cKit intensity. L) Statistical analysis of G for AβPP phosphorylation. Results are mean ± standard deviation, n=3. Asterisks (*) indicate statistical significance of p<0.05 by t-test.

Article Snippet: Equal amounts of protein from each sample were used for immunoprecipitation using V5-beads or anti-AβPP mAb 6E10, 22C11 (Sigma, St Louis) or mAb 4.1 (a gift from W. Van Nostrand) followed by immunoprecipitation with protein G agarose and western blotting with phosphotyrosine antibody (Cell Signaling, #9416). cKit and Shp2 knockdown with siRNA For cKit and Shp2 siRNA knockdown experiments, either scrambled negative control siRNA, cKit or Shp2 specific siRNA (Origene) were transfected in Opti-MEM with TransIT-X2 (Mirus) according to the manufacturer’s protocol overnight.

Techniques: Phospho-proteomics, Plasmid Preparation, Transfection, Inhibition, Residue, Stable Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Control, Standard Deviation

Journal: Journal of Alzheimer's disease : JAD

Article Title: Small Molecule Amyloid-β Protein Precursor Processing Modulators Lower Amyloid-β Peptide Levels via cKit Signaling

doi: 10.3233/JAD-180923

Figure Lengend Snippet: Kinetics and dose-response experiments for Shp2 inhibitor PHPS1-induced AβPP phosphorylation in HEK cells stably expressing AβPP751. A) Phosphorylation kinetics. IP-WB analysis of the effects of PHPS1 at 1 μM on AβPP phosphorylation at various time points. B) Dose-response experiments. IP-WB analysis of the effects of PHPS1 on AβPP phosphorylation at various concentrations at 2 h time point. All results are mean ± standard deviation, n=3. Asterisks (*) indicate statistical significance of p<0.05 by one-way ANOVA followed by Tukey post hoc test.

Article Snippet: Equal amounts of protein from each sample were used for immunoprecipitation using V5-beads or anti-AβPP mAb 6E10, 22C11 (Sigma, St Louis) or mAb 4.1 (a gift from W. Van Nostrand) followed by immunoprecipitation with protein G agarose and western blotting with phosphotyrosine antibody (Cell Signaling, #9416). cKit and Shp2 knockdown with siRNA For cKit and Shp2 siRNA knockdown experiments, either scrambled negative control siRNA, cKit or Shp2 specific siRNA (Origene) were transfected in Opti-MEM with TransIT-X2 (Mirus) according to the manufacturer’s protocol overnight.

Techniques: Phospho-proteomics, Stable Transfection, Expressing, Standard Deviation

Journal: Journal of Alzheimer's disease : JAD

Article Title: Small Molecule Amyloid-β Protein Precursor Processing Modulators Lower Amyloid-β Peptide Levels via cKit Signaling

doi: 10.3233/JAD-180923

Figure Lengend Snippet: Shp2 inhibitors enhance AβPP phosphorylation and reduce Aβ production. A) IP-WB analysis of the effects of two Shp2 inhibitors at 1 μM for 2 h on AβPP phosphorylation in AβPP stable cells. B) Two Shp-2 inhibitors as indicated at 1 μM for 18 h were tested for effects on Aβ40 level as measured by ELISA. Results are normalized to total AβPP. Results are mean ± standard deviation, n=3. Asterisks (*) indicate statistical significance of p<0.05 by t-test. C) Y10 and Shp2 inhibitors at 1 μM for 2 h also increase AβPP phosphorylation in primary mouse hippocampal neurons. D) Shp2 siRNA knockdown enhances AβPP phosphorylation. IP-WB analysis of the effect of control (Ctrl) or Shp2 siRNA on AβPP phosphorylation. E) Statistical analysis of AβPP phosphorylation in (D). Results normalized to AβPP input. p-Y, phosphotyrosine. F) Statistical analysis of Shp2 protein intensity in (D). All results are mean ± standard deviation, n=3. Asterisks (*) indicate statistical significance of p<0.05 by t-test. G) The effect of control (Ctrl), cKit siRNA, or Shp2 siRNA on Aβ40 level as measured by ELISA. Media collected 24-48 h post-transfection. Results are normalized to total AβPP. Results are mean ± standard deviation, n=3. Asterisks (**) indicate statistical significance of p<0.05 by t-test.

Article Snippet: Equal amounts of protein from each sample were used for immunoprecipitation using V5-beads or anti-AβPP mAb 6E10, 22C11 (Sigma, St Louis) or mAb 4.1 (a gift from W. Van Nostrand) followed by immunoprecipitation with protein G agarose and western blotting with phosphotyrosine antibody (Cell Signaling, #9416). cKit and Shp2 knockdown with siRNA For cKit and Shp2 siRNA knockdown experiments, either scrambled negative control siRNA, cKit or Shp2 specific siRNA (Origene) were transfected in Opti-MEM with TransIT-X2 (Mirus) according to the manufacturer’s protocol overnight.

Techniques: Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Standard Deviation, Knockdown, Control, Transfection

Journal: Journal of Alzheimer's disease : JAD

Article Title: Small Molecule Amyloid-β Protein Precursor Processing Modulators Lower Amyloid-β Peptide Levels via cKit Signaling

doi: 10.3233/JAD-180923

Figure Lengend Snippet: Shp2 siRNA knockdown enhances AβPP phosphorylation on Y743. A) IP-WB analysis of the effects of Shp2 siRNA on AβPP phosphorylation of WT AβPP751 or AβPP751 mutants (Y709G, Y738G, and Y743G) on each tyrosine residue in the C-terminal domain of transfected HEK293 cells. B, C) Statistical analysis of (A) showing Shp2 knockdown (B) and increased AβPP phosphorylation (C). Results normalized to Actin for Shp2 and to AβPP input for AβPP phosphorylation. p-Y, phosphotyrosine. Results are mean ± standard deviation, n=3. Asterisks (*) indicate statistical significance of p<0.05 by t-test.

Article Snippet: Equal amounts of protein from each sample were used for immunoprecipitation using V5-beads or anti-AβPP mAb 6E10, 22C11 (Sigma, St Louis) or mAb 4.1 (a gift from W. Van Nostrand) followed by immunoprecipitation with protein G agarose and western blotting with phosphotyrosine antibody (Cell Signaling, #9416). cKit and Shp2 knockdown with siRNA For cKit and Shp2 siRNA knockdown experiments, either scrambled negative control siRNA, cKit or Shp2 specific siRNA (Origene) were transfected in Opti-MEM with TransIT-X2 (Mirus) according to the manufacturer’s protocol overnight.

Techniques: Knockdown, Phospho-proteomics, Residue, Transfection, Standard Deviation

Journal: Journal of Alzheimer's disease : JAD

Article Title: Small Molecule Amyloid-β Protein Precursor Processing Modulators Lower Amyloid-β Peptide Levels via cKit Signaling

doi: 10.3233/JAD-180923

Figure Lengend Snippet: cKit and Shp2 inhibitors have no effect on BACE activity but enhance AβPP surface localization. A) BACE activity assay of cKit and Shp2 inhibitors. A BACE substrate analog peptide (0.1 μM) was used as inhibition control. B) The HEK293 cells stably expressing AβPP751 were treated with 1 μM of DMSO control, Y10 or PHPS1 for 2 h, and surface biotinylation assay was performed to analyze AβPP and a control membrane protein CD71 (transferrin receptor) surface localization. Biotinylated proteins were pulled-down by Neutravidin beads and detected by WB with anti-AβPP antibody (6E10), and anti-CD71 antibody. C) Statistical analysis of AβPP surface localization in (B). Results normalized to AβPP and CD71 inputs. p-Y, phosphotyrosine. Results are mean ± standard deviation, n=3. Asterisks (*) indicate statistical significance of p<0.05 by t-test.

Article Snippet: Equal amounts of protein from each sample were used for immunoprecipitation using V5-beads or anti-AβPP mAb 6E10, 22C11 (Sigma, St Louis) or mAb 4.1 (a gift from W. Van Nostrand) followed by immunoprecipitation with protein G agarose and western blotting with phosphotyrosine antibody (Cell Signaling, #9416). cKit and Shp2 knockdown with siRNA For cKit and Shp2 siRNA knockdown experiments, either scrambled negative control siRNA, cKit or Shp2 specific siRNA (Origene) were transfected in Opti-MEM with TransIT-X2 (Mirus) according to the manufacturer’s protocol overnight.

Techniques: Activity Assay, Inhibition, Control, Stable Transfection, Expressing, Surface Biotinylation Assay, Membrane, Standard Deviation

Journal: Journal of Alzheimer's disease : JAD

Article Title: Small Molecule Amyloid-β Protein Precursor Processing Modulators Lower Amyloid-β Peptide Levels via cKit Signaling

doi: 10.3233/JAD-180923

Figure Lengend Snippet: Dose response curve of cKit and Shp2 inhibitors on Aβ40 production. Concentration response curve of cKit inhibitors (PKC412, and AB1010) and Shp2 inhibitors (NSC87877 and PHPS1) on Aβ40 production as measured by ELISA in HEK293 cells stably overexpressing AβPP751. N=4 for each dose. Results were normalized to values obtained from cell viability assay by crystal violet staining. AβPP protein level is found to be linearly correlated to crystal violet staining (not shown). Results were normalized to % DMSO control.

Article Snippet: Equal amounts of protein from each sample were used for immunoprecipitation using V5-beads or anti-AβPP mAb 6E10, 22C11 (Sigma, St Louis) or mAb 4.1 (a gift from W. Van Nostrand) followed by immunoprecipitation with protein G agarose and western blotting with phosphotyrosine antibody (Cell Signaling, #9416). cKit and Shp2 knockdown with siRNA For cKit and Shp2 siRNA knockdown experiments, either scrambled negative control siRNA, cKit or Shp2 specific siRNA (Origene) were transfected in Opti-MEM with TransIT-X2 (Mirus) according to the manufacturer’s protocol overnight.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Stable Transfection, Viability Assay, Staining, Control

Journal: Journal of Alzheimer's disease : JAD

Article Title: Small Molecule Amyloid-β Protein Precursor Processing Modulators Lower Amyloid-β Peptide Levels via cKit Signaling

doi: 10.3233/JAD-180923

Figure Lengend Snippet: Current model for potential interactions among AβPP, cKit, Shp2, and Gab2. Gab2 is a scaffold protein found by GWAS to be associated with AD. Inhibition of cKit and Shp2 increases AβPP phosphorylation on the Y743 residue resulting in a reduction in Aβ production. Kinase and phosphatase inhibitors that increased AβPP phosphorylation and reduced Aβ formation are in pink boxes. Yellow P, pSer; Green P, pTyr; SCF, stem cell factor.

Article Snippet: Equal amounts of protein from each sample were used for immunoprecipitation using V5-beads or anti-AβPP mAb 6E10, 22C11 (Sigma, St Louis) or mAb 4.1 (a gift from W. Van Nostrand) followed by immunoprecipitation with protein G agarose and western blotting with phosphotyrosine antibody (Cell Signaling, #9416). cKit and Shp2 knockdown with siRNA For cKit and Shp2 siRNA knockdown experiments, either scrambled negative control siRNA, cKit or Shp2 specific siRNA (Origene) were transfected in Opti-MEM with TransIT-X2 (Mirus) according to the manufacturer’s protocol overnight.

Techniques: Inhibition, Phospho-proteomics, Residue