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 , A schematic representation of the possible conformations for the ITGB1 intramolecular FRET biosensor (Illusia), where the mTurquoise2–YPet FRET pair is separated by the PTB from Dok1, a linker and the cytoplasmic domain (aa772–798) from ITGB1 (including the two NPxY motifs). Illusia is recruited to the membrane through an acylation substrate sequence derived from Lyn kinase. Ex, excitation; Em, emission. b , Representative FLIM images (left) and quantification of apparent FRET efficiency (right) of MM231 cells stably expressing Illusia after Dox-inducible overexpression of either Src(WT), kinase-dead Src(K295R) or constitutively active Src(Y527F)/Src(E378G) ( n = 60 cells in all conditions with the exception of 70 for Src(Y527F) (−)Dox and 65 for Src(Y527F) (+)Dox pooled from three biological replicates; one-way analysis of variance (ANOVA) with a Šidák correction for multiple comparisons). Scale bars, 10 μm. c , Representative western blot of MM231 cells after Dox-inducible Src(WT), Src(K295R), Src(Y527F) or Src(E378G) overexpression. d , Densitometry analysis of western blots from c ( n = 4 biological replicates; one-sample two-tailed t -test against the normalized control values for each cell line without Dox). The data are mean ± s.e.m. e , A scheme of an ELISA for pY. f , An ELISA for changes in ITGB1 phosphorylation using recombinant ITGB1 peptide and Src kinase in the absence or presence of ATP and the Src inhibitor Sara (1 μM; n = 3 biological replicates; triplicate wells/replicates; one-way ANOVA with a Šidák correction for multiple comparisons). Unphosphorylated ITGB1 and phosphorylated ITGB1 p(Y783) peptides were included as negative and positive controls, respectively. The data are the mean ± s.e.m. g , Representative FLIM–FRET images (left) and quantification (right) of MM231 and TIF cells with stable expression of Illusia and Sara treatment (1 μM) for 24 h (MM231s, n = 129 (DMSO) and 125 (Sara) cells pooled from five biological replicates; TIFs, n = 74 (DMSO) and 75 (Sara) cells pooled from three biological replicates; unpaired two-tailed Student’s t -test with a Welch’s correction). Scale bars, 10 μm. The boxplots represent the median and IQR. The whiskers extend to the minimum and maximum values. The grey areas on the boxplots highlight the IQR of the control conditions. NS, not significant. * P < 0.05, *** P < 0.001. .

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: Membrane, Sequencing, Derivative Assay, Stable Transfection, Expressing, Over Expression, Western Blot, Two Tailed Test, Control, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Recombinant

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 western blots of MM231 and MCF10A cells expressing either Venus or Dok1 (n = 4 biological replicates). b , A schematic of the intermolecular FRET approach between Dok1 and ITGB1 (WT or YYFF). c , Representative FLIM-FRET images (left) and quantification of apparent FRET efficiency (right) from MM231 shβ1 cells reexpressing mRuby2-tagged ITGB1(WT or YYFF) and transfected with Dok1-Clover (n = 60 cells in each condition 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, 10 μm. d , Representative FLIM-FRET images (left) and quantification of apparent FRET efficiency (right) from MM231 shβ1 cells reexpressing mRuby2-tagged ITGB1(WT or YYFF), and transfected with GFP-F 0 F 3 talin head domain fragment (n = 62 cells in each condition 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. e & f , Apparent FRET efficiencies ( e ) and representative images ( f ) of MM231 cells stably expressing Illusia and Dox-inducible ABL2(WT) or kinase-dead ABL2(K281M) ± Dox treatment. Parental cells treated with Dox are used as an additional control (n = 60 cells in each condition 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, 10 μm. g & h , Representative western blot ( g ) and densitometry ( h ) analysis of ITGB1 phosphorylation levels in MM231 cells with Dox-induced ABL2(WT) or ABL2(K281M) overexpression (n = 5 biological replicates; significance assessed using a one-sample two-tailed t -test against the normalised control values for each cell line without Dox; NS, not significant, * p < 0.05). Data are mean ± SEM. i & j , Representative western blot ( i ) and densitometry ( j ) of MM231 cells treated with saracatenib (Sara) for 0, 2, 24, 48 and 72 h (1 μM; n = 5 biological replicates; significance assessed using a one-sample two-tailed t -test against the normalised DMSO control). 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 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, Expressing, Transfection, Two Tailed Test, Stable Transfection, Control, Phospho-proteomics, Over Expression

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 , 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.

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: Phospho-proteomics, Western Blot, Two Tailed Test, Control, Stable Transfection, Expressing, Mutagenesis

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 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.

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: Transfection, Two Tailed Test, Staining, Control

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 , A schematic of the interaction between ITGB1-V1 and a V2-tagged adaptor or regulator, highlighting the resulting V1/V2 (Venus) protein complex as the restored epitope for BiCAP (top) or donor for BiFC–FLIM–FRET (bottom). The Venus tag alone was used as a control for the BiCAP and BiFC–FLIM–FRET experiments. b , A representative BiCAP immunoblot after HEK293T cell transfection with either Venus or ITGB1-V1/Dok1-V2 ( n = 3 biological replicates). c , Representative BiCAP immunoblots from MM231 and HEK293T cells where annexin A6 (AnxA6), VPS35 and Cofilin coimmunoprecipitate with the Dok1/ITGB1 complex ( n = 3 biological replicates). d , Representative BiFC–FLIM–FRET images (left) and quantification of apparent FRET efficiency (right) from MM231 cells transfected with RFP-tagged Cofilin mutants WT, S3A and S3E (Venus, n = 73 (WT), 72 (S3A) and 64 (S3E); BiFC, n = 92 (WT), 64 (S3A) and 64 (S3E) cells from three biological replicates; one-way analysis of variance (ANOVA) with a Tukey correction for multiple comparisons). Scale bars, 20 μm. e , Representative images (left) and quantification of gelatin degradation (right) by MM231 ITGB1(WT or YYFF) cells with Dox-inducible Src(E378G) expression. The MM231 cells were transfected with siRNAs against Dok1 (siDok1_1 and siDok1_2) or a NTC siRNA and treated with Dox for 24 h before being seeded on fluorescent gelatin (green) for 6 h (actin labelled with SiR-actin (white), nuclei with DAPI (blue)) (ITGB1(WT), n = 36 (NTC), 38 (siDok1_1) and 36 (siDok1_2); ITGB1(YYFF), n = 38 (NTC), 34 (siDok1_1) and 37 (siDok1_2) fields of view pooled from three biological replicates; one-way ANOVA with a Šidák correction for multiple comparisons). Scale bars, 20 μm; insets, 10 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 < 0.001. .

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: Control, Western Blot, Transfection, Expressing

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 – d , Representative images (left) and quantification of apparent FRET efficiency (right) for intermolecular FLIM–FRET of the following tagged protein pairs, Dok1–Clover/Cofilin–mRFP ( a ), CTTN–mEmerald/Dok1–mScarlet ( b ), Dok1–Clover/mScarlet–TKS5 ( c ) and CTTN–mEmerald/Cofilin–mRFP ( d ). FRET between mScarlet and the donor-tagged protein was used as a negative control for all pairs (for a , n = 85 (Dok1/mScarlet) and 95 (Dok1/Cofilin) cells pooled from five biological replicates; for b , n = 65 (CTTN/mScarlet) and 68 (CTTN/Dok1) cells pooled from three biological replicates; for c , n = 62 cells for each condition pooled from three biological replicates; for d , n = 65 (CTTN/mScarlet) and 70 (CTTN/Cofilin) cells pooled from three biological replicates; unpaired two-tailed Student’s t -test with a Welch’s correction). Scale bars, 20 μm. e , Representative images of mice with MM231 ITGB1(WT or YYFF) cells stably expressing the luciferase/EGFP construct. Oral gavage of Vehicle or SHP099 (100 mg kg −1 ) proceeded for 5 days from the day of injection. f , A box and whisker plot highlighting the endpoint metastatic burden as an average (Avg) radiance value from the luciferase signal of the MM231 cells in e ( n = 9 mice tracked per group). g , Representative lung sections stained for EGFP-positive MM231 cells. Scale bars, 2 mm; insets: 200 μm. h , Quantification of pulmonary nodule number (that is, clusters of greater than ten cells) in lungs from EGFP-positive MM231 cells ( n = 10 mice per group). i , Quantitative real-time PCR of the RNA samples collected from the MM231 ITGB1(WT or YYFF) cells stably expressing the luciferase/EGFP construct. The mice were designated as either ‘metastatic’ or ‘low signal’ after setting a threshold for ‘metastatic’ as having an expression fold change >1 compared with the mean of the WT/vehicle control with human GAPDH normalized to mouse/human GAPDH ( n = 10 mice/group). The boxplots represent the median and IQR. The whiskers extend to min and max values. The grey areas highlight the IQR of the control conditions. *** P < 0.001. .

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: Negative Control, Two Tailed Test, Stable Transfection, Expressing, Luciferase, Construct, Injection, Whisker Assay, Staining, Real-time Polymerase Chain Reaction, Control

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