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: a , Schematic of the FRET experiment (left), with representative FLIM-FRET images (middle) and quantification of apparent FRET efficiency (right) of MM231 cells with stable expression of either ITGB1(WT)-mRuby2 or ITGB1(YYFF)-mRuby2 transfected with Clover and treated with VO 4 3- (n = 65 [ITGB1(WT) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(-){\rm{VO}}}_{4}^{3-}$$\end{document} ( − ) VO 4 3 − ], 70 [ITGB1(WT) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(+){\rm{VO}}}_{4}^{3-}$$\end{document} ( + ) VO 4 3 − ], 72 [ITGB1(YYFF) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(-){\rm{VO}}}_{4}^{3-}$$\end{document} ( − ) VO 4 3 − ], and 66 [ITGB1(YYFF) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(+){\rm{VO}}}_{4}^{3-}$$\end{document} ( + ) VO 4 3 − ]) cells pooled from three biological replicates. Scale bars, 20 μm. b , Representative FLIM-FRET images (left) and quantification of apparent FRET efficiency (right) in MM231 cells with stable Illusia expression and constitutive overexpression of PTPN11 (Shp2, WT) or a phosphatase-dead mutant (Shp2, Mut; PTPN11(D425A, C459S)) (n = 97 (mScarlet), 96 (WT) and 96 (Mut) cells pooled from four biological replicates; significance assessed using a one-way ANOVA with a Tukey correction for multiple comparisons; NS, not significant, *** p < 0.001). Scale bars, 20 μm. c , Representative western blot (left) and densitometry (right) of MM231 cells with stable Illusia expression and constitutive overexpression of PTPN11 (Shp2, WT) or a phosphatase-dead mutant (Shp2, Mut; PTPN11(D425A, C459S); n = 8 biological replicates; significance assessed using a one-sample two-tailed t -test against the normalised control value; * p < 0.05, ***p < 0.001; NS, not significant). d-e , Representative FLIM-FRET images ( d ) and quantification of apparent FRET efficiency ( e ) of MM231 cells with stable Illusia expression treated overnight with Dox to induce overexpression of PTP-PEST WT or a phosphatase-dead mutant (Mut, PTPN12(D199A, C231S) (n = 100 cells in each condition pooled from four biological replicates; significance assessed using a one-way ANOVA with a Šidák correction for multiple comparisons; NS, not significant, *** p < 0.001). Scale bars, 20 μm. f , Western blot (left) and densitometry (right) from parallel data in ( d ; n = 4 biological replicates; significance assessed using a one-sample two-tailed t -test against the normalised control value; * p < 0.05; NS, not significant). g & h , Representative FLIM-FRET images (left) and quantification of apparent FRET efficiency (right) of MM231 ( g ) and TIF ( h ) cells with stable Illusia expression and treated with SHP099 for 2 h (100 nM) (MM231, n = 96 (DMSO) and 95 (SHP099) | TIFs, n = 100 (DMSO) and 99 (SHP099) cells pooled from four biological replicates; significance assessed using an unpaired two-tailed Student’s t -test with a Welch’s correction (NS, not significant, *** p < 0.001). Scale bars, 20 μm. i , Representative western blots (left) and densitometry (right) of MM231 and TIF cells with stable Illusia expression and treated with SHP099 for 2 h (100 nM; n = 6 biological replicates; significance assessed using a one-sample two-tailed t -test against the normalised control value; * p < 0.05). Data are mean ± SEM. Source data and exact p-values are provided in the statistical source data file. Boxplots represent median and interquartile range. Whiskers extend to min and max values. Grey areas on boxplots highlight the interquartile range of the control conditions.
Article Snippet: The primary antibodies against ITGB1 (1:1,000, Abcam, ab52971), ITGB1(Y783) (1:500, Abcam, ab62337), PTP-PEST (1:1,000, Cell Signalling, 14735), Shp2 (1:1,000, Cell Signalling, 3397), Src (1:1,000, Cell Signalling, 2108), Src(Y416) (1:500, Cell Signalling, 2101), Arg (1:1,000, Abcam, ab134134), p130Cas (1:1,000, Santa Cruz, sc-20029), p130Cas(Y165) (1:1,000, Cell Signalling, 4015), p130Cas(Y410) (1:1,000, Cell Signalling, 4011), pY (1:1,000, BD Biosciences, 610000), GFP (1:1,000, Thermo Fisher, A11122), RFP (1:1,000; Chromotek, 6g6-100), Dok1 (1:1,000, Abcam, ab8112), VPS35 (1:1,000, Abcam, ab10099), Cofilin (D3F9; 1:1,000, Cell Signalling, 5175), Tks5 (1:500, Millipore, MABT336), β-Actin (1:10,000, Sigma, A1978), Cortactin (CTTN; clone 4F11; 1:500, Millipore, 05-180), annexin A6 (1:500, Abcam, ab31026) and GAPDH (1:10,000; Hytest, 5G4MAB6C5) were incubated overnight at 4 °C in AdvanBlock-Fluor.
Techniques: Expressing, Transfection, Over Expression, Mutagenesis, Western Blot, Two Tailed Test, Control
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![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 transfected with either Shp2–Clover ( c ) or PTP-PEST-Clover ( d ) and treated with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\rm{VO}}}_{4}^{3-}$$\end{document} VO 4 3 − (100 μM, 2 h) (for c , n = 73 (ITGB1(WT) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(-){\rm{VO}}}_{4}^{3-}$$\end{document} ( − ) VO 4 3 − ), 62 (ITGB1(WT) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(+){\rm{VO}}}_{4}^{3-}$$\end{document} ( + ) VO 4 3 − ), 64 (ITGB1(YYFF) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(-){\rm{VO}}}_{4}^{3-}$$\end{document} ( − ) VO 4 3 − ) and 67 (ITBG1(YYFF) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(+){\rm{VO}}}_{4}^{3-}$$\end{document} ( + ) VO 4 3 − ) cells pooled from three biological replicates; for d , n = 75 (ITGB1(WT) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(-){\rm{VO}}}_{4}^{3-}$$\end{document} ( − ) VO 4 3 − ), 58 (ITGB1(WT) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(+){\rm{VO}}}_{4}^{3-}$$\end{document} ( + ) VO 4 3 − ), 58 (ITGB1(YYFF) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(-){\rm{VO}}}_{4}^{3-}$$\end{document} ( − ) VO 4 3 − ) and 65 (ITBG1(YYFF) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(+){\rm{VO}}}_{4}^{3-}$$\end{document} ( + ) VO 4 3 − ) cells pooled from three biological replicates; one-way analysis of variance with a Tukey correction for multiple comparisons). Scale bars, 20 μm. The boxplots represent the median and IQR. The whiskers extend to the minimum and maximum values. The grey areas highlight the IQR of the control conditions. NS, not significant. * P < 0.05, ** P < 0.01, *** P < 0.001. .](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_3946/pmc12173946/pmc12173946__41556_2025_1663_Fig5_HTML.jpg)
![a , Schematic of the FRET experiment (left), with representative FLIM-FRET images (middle) and quantification of apparent FRET efficiency (right) of MM231 cells with stable expression of either ITGB1(WT)-mRuby2 or ITGB1(YYFF)-mRuby2 transfected with Clover and treated with VO 4 3- (n = 65 [ITGB1(WT) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(-){\rm{VO}}}_{4}^{3-}$$\end{document} ( − ) VO 4 3 − ], 70 [ITGB1(WT) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(+){\rm{VO}}}_{4}^{3-}$$\end{document} ( + ) VO 4 3 − ], 72 [ITGB1(YYFF) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(-){\rm{VO}}}_{4}^{3-}$$\end{document} ( − ) VO 4 3 − ], and 66 [ITGB1(YYFF) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(+){\rm{VO}}}_{4}^{3-}$$\end{document} ( + ) VO 4 3 − ]) cells pooled from three biological replicates. Scale bars, 20 μm. b , Representative FLIM-FRET images (left) and quantification of apparent FRET efficiency (right) in MM231 cells with stable Illusia expression and constitutive overexpression of PTPN11 (Shp2, WT) or a phosphatase-dead mutant (Shp2, Mut; PTPN11(D425A, C459S)) (n = 97 (mScarlet), 96 (WT) and 96 (Mut) cells pooled from four biological replicates; significance assessed using a one-way ANOVA with a Tukey correction for multiple comparisons; NS, not significant, *** p < 0.001). Scale bars, 20 μm. c , Representative western blot (left) and densitometry (right) of MM231 cells with stable Illusia expression and constitutive overexpression of PTPN11 (Shp2, WT) or a phosphatase-dead mutant (Shp2, Mut; PTPN11(D425A, C459S); n = 8 biological replicates; significance assessed using a one-sample two-tailed t -test against the normalised control value; * p < 0.05, ***p < 0.001; NS, not significant). d-e , Representative FLIM-FRET images ( d ) and quantification of apparent FRET efficiency ( e ) of MM231 cells with stable Illusia expression treated overnight with Dox to induce overexpression of PTP-PEST WT or a phosphatase-dead mutant (Mut, PTPN12(D199A, C231S) (n = 100 cells in each condition pooled from four biological replicates; significance assessed using a one-way ANOVA with a Šidák correction for multiple comparisons; NS, not significant, *** p < 0.001). Scale bars, 20 μm. f , Western blot (left) and densitometry (right) from parallel data in ( d ; n = 4 biological replicates; significance assessed using a one-sample two-tailed t -test against the normalised control value; * p < 0.05; NS, not significant). g & h , Representative FLIM-FRET images (left) and quantification of apparent FRET efficiency (right) of MM231 ( g ) and TIF ( h ) cells with stable Illusia expression and treated with SHP099 for 2 h (100 nM) (MM231, n = 96 (DMSO) and 95 (SHP099) | TIFs, n = 100 (DMSO) and 99 (SHP099) cells pooled from four biological replicates; significance assessed using an unpaired two-tailed Student’s t -test with a Welch’s correction (NS, not significant, *** p < 0.001). Scale bars, 20 μm. i , Representative western blots (left) and densitometry (right) of MM231 and TIF cells with stable Illusia expression and treated with SHP099 for 2 h (100 nM; n = 6 biological replicates; significance assessed using a one-sample two-tailed t -test against the normalised control value; * p < 0.05). Data are mean ± SEM. Source data and exact p-values are provided in the statistical source data file. Boxplots represent median and interquartile range. Whiskers extend to min and max values. Grey areas on boxplots highlight the interquartile range of the control conditions.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_3946/pmc12173946/pmc12173946__41556_2025_1663_Fig13_ESM.jpg)