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 , Representative immunoblot from MM231 ITGB1 (WT or YYFF) cells and doxycycline (Dox)-inducible Src(E378G) transfected with siRNAs against Dok1, treated with Dox for 24 h and blotted for Cofilin, TKS5, CTTN and Dok1. b , X-Y correlation plots from densitometric analysis of western blots in ( a ), normalising to the actin loading control for Cofilin, TKS5, CTTN and Dok1. Plotted are graphs showing correlations between Dok1-Cofilin (left), Dok1-TKS5 (middle) and Dok1-CTTN (right; n = 7 biological replicates; significance assessed using an X-Y linear regression). c , qPCR data from MM231 cells transfected with siRNAs against DOK1 (siDok1_1 and siDok1_2), or a NTC (siNTC), and treated with Dox for 24 h before processing for qRT-PCR. Plotted is the relative mRNA fold-change, normalised to GAPDH (n = 4 biological replicates, performed in duplicate or triplicate; significance assessed using a one sample t -test against the normalised siNTC control values; NS, not significant, *** p < 0.001, ** p < 0.01, * p < 0.05). d , Schematic of luciferase/EGFP construct (top) and qRT-PCR (bottom) for EGFP fold-change between the MM231 ITGB1(WT or YYFF) cells stably-expressing the luciferase/EGFP construct, normalised to GAPDH (n = 4 biological replicates, performed in triplicate; significance assessed using a one-sample two-tailed t -test against the normalised control values; NS, not significant). e , Representative images of mice immediately after lateral tail vein injection with MM231 ITGB1(WT or YYFF) cells stably-expressing the luciferase/EGFP construct and pre-treated with either DMSO or SHP099 (100 nM). Oral gavage of Vehicle or SHP099 (100 mg/kg) proceeded for 5 days from the day of injection. f , Quantification of the average (avg) radiance from the luciferase signal during the colonisation stages of the MM231 cells (n = 9 mice tracked/group; mean ± SEM). g , Quantification of metastatic area (%) in lungs from EGFP-positive MM231 cells (n = 10 mice/group; scatter plot with a line at the median value). h , Assessment of the ratio of micro- to macrometastasis in lungs from clusters of EGFP-positive MM231 cells lesser or greater than 3,000 µm 2 respectively; excluding mice where no micro- or macrometastasis were detected (n = 9, 8 & 7 mice for the WT/vehicle, WT/SHP099 and YYFF/vehicle groups respectively; scatter plot with a line at the median value). i & j , Gating strategy ( i ) and flow cytometric analysis ( j ) of annexinV-stained MM231s with ITGB1(WT or YYFF) and treated with either DMSO or SHP099 (SHP; 100 nM) while grown overnight in suspension on ultra-low attachment plates and either subjected to flow and/or treated with SHP099 (100 nM) or DMSO control (negative (-ve): unstained negative control samples; positive ( + ve): MM231s treated with a cell-death-inducing cocktail of doxorubicin (10 µg/ml), \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 − (50 µM), gemcitabine (10 µM); n = 4 biological replicates/cell line for DMSO and SHP099 “No flow” and n = 3 biological replicates for all other conditions; mean ± SEM; Statistics from a one-way ANOVA with a Tukey correction for multiple comparisons; NS, not significant). 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.
Article Snippet: The following Taqman gene expression assays were used: Colfilin-2/ CFL2 (Hs01071313_g1), Cofilin-1/ CFL1 (Hs02621564_g1), Cortactin/ CTTN (Hs01124232_m1), Tks5 ( SH3PXD2A , (Hs01046307_m1), Dok1 ( DOK1 , Hs00796733_s1), EGFP (Mr04329676_mr), receptor-type tyrosine-protein phosphatase-like N/ PTPRN (Hs00160947_m1), PTP-PEST/ PTPN12 (Hs00184747_m1), myotubularin-related protein 8/ MTMR8 (Hs00250307_m1), cell division cycle 14B/ CDC14B (Hs00269351_m1), transmembrane phosphatase with tensin homology/ TPTE (Hs00276201_m1), myotubularin/ MTM1 (Hs00896975_m1), PTP non-receptor-type 20/ PTPN20 (Hs00944181_m1), receptor-type tyrosine-protein phosphatase O/ PTPRO (Hs00958177_m1), Slingshot homologue 2/ SSH2 (Hs00987189_m1), dual-specificity phosphatase 8/ DUSP8 (Hs01014943_m1), dual-specificity phosphatase 16/ DUSP16 (Hs01015508_m1), dual-specificity phosphatase 3/ DUSP3 (Hs01115776_m1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/ GAPDH (Hs02786624_g1).
Techniques: Western Blot, Transfection, Control, Quantitative RT-PCR, Luciferase, Construct, Stable Transfection, Expressing, Two Tailed Test, Injection, Staining, Suspension, Negative Control
Santa Cruz Biotechnology is a verified supplier 
![a , A schematic of phosphorylation-dependent Dok1 recruitment to ITGB1. b , c , Representative western blots ( b ) and densitometry ( c ) of MM231 (left) or MCF10A (right) cells treated with the broad-spectrum PTP inhibitor sodium orthovanadate ( \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; n = 4 biological replicates; one-sample two-tailed t -test against the normalized control value without \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 − ). The data are the mean ± s.e.m. d , A representative western blot (left) of MM231 ITGB1(WT or YYFF) cells treated for 2 h 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) and densitometry analysis (right) of MM231 ITGB1(WT) cells ( n = 5 biological replicates; one-sample two-tailed t -test against the normalized control value without \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 − ). The data are the mean ± s.e.m. e , Representative FLIM images (left) and quantification of apparent FRET efficiency (right) after \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 − treatment (100 μM, 2 h) of MM231 cells stably expressing Illusia(WT) or a non-phosphorylatable mutant reporter Illusia(YYFF) ( n = 88 (Illusia(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 − )), 88 (Illusia(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 − )), 93 (Illusia(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 86 (Illusia(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 four biological replicates; one-way analysis of variance with a Šidák correction for multiple comparisons). n.s., not significant. Scale bars, 10 μm. The boxplot represents the median and IQR. The whiskers extend to the minimum and maximum values. The grey area on the boxplot highlights the IQR of the control condition. NS, not significant, * P < 0.05, *** P < 0.001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_3946/pmc12173946/pmc12173946__41556_2025_1663_Fig3_HTML.jpg)
![a , Representative BiFC-FLIM-FRET images (left) and quantification of apparent FRET efficiency (right) from MM231 cells transfected with Venus or ITGB1-V1/ Dok1-V2 and either mScarlet-tagged Annexin A6 or VPS35 (n = 61 for each Annexin A6 condition, 58 (VPS35, Venus) and 60 (VPS35, BiFC) cells pooled from three biological replicates; significance assessed using an unpaired two-tailed Student’s t -test with a Welch’s correction; *** p < 0.001). Scale bars, 20 μm. b , Schematic of an invadopodium degrading the ECM. c , Representative images (left) and quantification (right) of MM231 ITGB1(WT or YYFF) cells with doxycycline(Dox)-inducible Src(E378G), treated (+/-)Dox overnight and then seeded on fluorescent gelatin (green) for 6 h (white, SiR-Actin stain; blue, DAPI nuclear stain; n = 30 [ITGB1 (WT) (-)Dox], 41 [ITGB1 (WT) ( + )Dox], 34 [ITGB1(YYFF) (-)Dox] and 41 [ITGB1(YYFF) ( + )Dox] fields of view pooled from three 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. 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_Fig15_ESM.jpg)
![a , Representative immunoblot from MM231 ITGB1 (WT or YYFF) cells and doxycycline (Dox)-inducible Src(E378G) transfected with siRNAs against Dok1, treated with Dox for 24 h and blotted for Cofilin, TKS5, CTTN and Dok1. b , X-Y correlation plots from densitometric analysis of western blots in ( a ), normalising to the actin loading control for Cofilin, TKS5, CTTN and Dok1. Plotted are graphs showing correlations between Dok1-Cofilin (left), Dok1-TKS5 (middle) and Dok1-CTTN (right; n = 7 biological replicates; significance assessed using an X-Y linear regression). c , qPCR data from MM231 cells transfected with siRNAs against DOK1 (siDok1_1 and siDok1_2), or a NTC (siNTC), and treated with Dox for 24 h before processing for qRT-PCR. Plotted is the relative mRNA fold-change, normalised to GAPDH (n = 4 biological replicates, performed in duplicate or triplicate; significance assessed using a one sample t -test against the normalised siNTC control values; NS, not significant, *** p < 0.001, ** p < 0.01, * p < 0.05). d , Schematic of luciferase/EGFP construct (top) and qRT-PCR (bottom) for EGFP fold-change between the MM231 ITGB1(WT or YYFF) cells stably-expressing the luciferase/EGFP construct, normalised to GAPDH (n = 4 biological replicates, performed in triplicate; significance assessed using a one-sample two-tailed t -test against the normalised control values; NS, not significant). e , Representative images of mice immediately after lateral tail vein injection with MM231 ITGB1(WT or YYFF) cells stably-expressing the luciferase/EGFP construct and pre-treated with either DMSO or SHP099 (100 nM). Oral gavage of Vehicle or SHP099 (100 mg/kg) proceeded for 5 days from the day of injection. f , Quantification of the average (avg) radiance from the luciferase signal during the colonisation stages of the MM231 cells (n = 9 mice tracked/group; mean ± SEM). g , Quantification of metastatic area (%) in lungs from EGFP-positive MM231 cells (n = 10 mice/group; scatter plot with a line at the median value). h , Assessment of the ratio of micro- to macrometastasis in lungs from clusters of EGFP-positive MM231 cells lesser or greater than 3,000 µm 2 respectively; excluding mice where no micro- or macrometastasis were detected (n = 9, 8 & 7 mice for the WT/vehicle, WT/SHP099 and YYFF/vehicle groups respectively; scatter plot with a line at the median value). i & j , Gating strategy ( i ) and flow cytometric analysis ( j ) of annexinV-stained MM231s with ITGB1(WT or YYFF) and treated with either DMSO or SHP099 (SHP; 100 nM) while grown overnight in suspension on ultra-low attachment plates and either subjected to flow and/or treated with SHP099 (100 nM) or DMSO control (negative (-ve): unstained negative control samples; positive ( + ve): MM231s treated with a cell-death-inducing cocktail of doxorubicin (10 µg/ml), \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 − (50 µM), gemcitabine (10 µM); n = 4 biological replicates/cell line for DMSO and SHP099 “No flow” and n = 3 biological replicates for all other conditions; mean ± SEM; Statistics from a one-way ANOVA with a Tukey correction for multiple comparisons; NS, not significant). 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.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_3946/pmc12173946/pmc12173946__41556_2025_1663_Fig16_ESM.jpg)