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