Journal: The EMBO Journal

Article Title: PTEN deficiency exposes a requirement for an ARF GTPase module for integrin‐dependent invasion in ovarian cancer

doi: 10.15252/embj.2023113987

Figure Lengend Snippet: A Schema, (1) CRISPR screen. 26 ARF6‐proximal proteins from TurboID studies were investigated for their contribution to ARF6‐mediated invasion of ID8 Trp53 −/− ; Pten −/− spheroids. (2) For each interactor, 5 sgRNAs were cloned into lentiviral CRISPR vectors. (3) A pooled approach was used, generating a KO cell line with all 5 sgRNAs (4) Live imaging performed. (5) Phenotype of each KO compared with nontargeting sgRNA. B Frequency of Spherical and Hyper‐protrusive phenotypes upon pooled gRNA CRISPR of indicated targets (sorted based on hierarchical clustering) in ID8 Trp53 −/− ; Pten −/− clone 1.15 cells, performed in four parts (Iterations indicated). Heatmap (grayscale)—phenotype proportion ( z ‐score) in control (sgNT). Heatmap (blue‐red)—log 2 fold change from control. P ‐values, bubble size (Cochran–Mantel–Haenszel test with Bonferroni adjustment). Black dot, homogenous effect across independent experiments (Breslow–Day test, Bonferroni adjustment, nonsignificant). N = 3–4 independent experiments, 3–6 technical replicates/experiment. Total spheroid number per condition, Table . C Western blot, β1‐integrin (ITGB1), pS473‐AKT, AKT, ARF6 from deconvolved ITGB1 sgRNA‐expressing cells. VCL, loading control for ITGB1, sample integrity control for other blots. Representative blots of n = 3 independent lysate preparations. D Quantitation of (C). Data, mean ± SD for pS473‐AKT:total AKT band intensity ratio, total AKT or ARF6 intensity, normalised to control (sgNT ID8 Trp53 −/− ; Pten −/− clone 1.15) cells. P ‐values, unpaired, two‐tailed t ‐test. E, F Frequency of Spherical and Hyper‐protrusive phenotypes in ID8 Trp53 −/− ; Pten −/− 1.15spheroids upon CRISPR‐mediated KO of (E) Itgβ1 or (F) Agap1 , 6 h time intervals over 72 h. Heatmap (grayscale)—phenotype proportion ( z ‐score) in control (sgNT). Heatmap (blue‐red)—log 2 fold change from control. P ‐values, bubble size (Cochran–Mantel–Haenszel test with Bonferroni adjustment). Black dot, homogenous effect across independent experiments (Breslow–Day test, Bonferroni adjustment, non‐significant). N = 3 independent experiments, 1–5 technical replicates/experiment. Total spheroid number per condition, Table . G Representative phase contrast images of spheroids described in (E, F). Outlines pseudocoloured for classification (Spherical, green; Hyper‐protrusive, blue) at indicated timepoints. Magnified individual spheroids from boxed regions. Arrowheads, protrusions into ECM. Scale bars, 400 or 17 μm, as indicated. H Representative confocal images of Trp53 −/− and Trp53 −/− ; Pten −/− clone 1.15 spheroids expressing sgNT, sg Agap1 (sg3) or sg Itgb1 (sg4), stained for collagen IV (grayscale) and F‐Actin (magenta). Boxed areas, basement membrane region in higher magnification. Arrowheads, Collagen IV labelling that is: well‐defined, green; fragmented, yellow; absent, navy. Scale bar, 53 μm. I Quantitation of (H). Collagen IV basement membrane staining as Defined, Fragmented, or Absent in spheroids set up across n = 3 independent experiments, 1 technical replicate/experiment, 5–9 fields imaged per technical replicate, 365 spheroids scored in total. Data, mean ± SD of % of spheroids in each phenotype for independent experiments, with circles representing technical replicates. Unpaired t ‐test, P ‐values annotated. Source data are available online for this figure.

Article Snippet: Antibodies used were: anti‐2A peptide (Merck, MABS2005, 3H4), anti‐AKT pan (CST, 2920, 40D4), anti‐ARF5 (Novus Biologicals, H0000281‐M01, IB4), anti‐ARF6 (Merck, A5230), anti‐GAPDH (CST 2118, 14C10, 1:5,000), anti‐α5 integrin (Abcam, ab150361), anti‐β1 integrin (Merck, clone MB1.2), anti‐AKT phospho S473 (CST, 4060, D9E), anti‐S6RP phospo S235/236 (CST, 2217, 5G10), anti‐S6RP (CST, 2217, 5G10), PTEN (CST, 9552), anti‐RFP (used to detect BFP; Thermo Fisher Scientific, R10367), anti‐Streptavidin‐Horseradish Peroxidase (HRP) Conjugated (Thermo Fisher Scientific, SA10001), anti‐TP53 (Abcam, ab26, diluted in 5% milk in TBS‐T), anti‐V5 Tag (ABM, G189), anti‐Vinculin (Merck, V9131, 1:2,000).

Techniques: CRISPR, Clone Assay, Imaging, Western Blot, Expressing, Quantitation Assay, Two Tailed Test, Staining, Membrane

Journal: The EMBO Journal

Article Title: PTEN deficiency exposes a requirement for an ARF GTPase module for integrin‐dependent invasion in ovarian cancer

doi: 10.15252/embj.2023113987

Figure Lengend Snippet: A, B Immunofluorescence and confocal imaging of Trp53 −/− ; Pten −/− 1.15 spheroids stained for α5‐integrin or β1‐integrin (grey or FIRE LUT), Hoechst (blue) and F‐actin (magenta). Magnified images from boxed regions shown. Arrowheads, labelling at protrusion tips. Scale bars, 5 μm. Representative of n = 3 spheroids imaged. (B) Intensity profiles for integrins (grey) and F‐actin (magenta) from spheroids in (A). Tip measured is annotated, ECM to body, yellow arrow, tip, white arrowhead. C, D Immunofluorescence and confocal imaging of Trp53 −/− ; Pten −/− 1.15 spheroids stained for pFAK (Y379) or pSRC Family Kinases (SFK pY416) (grey or FIRE LUT), Hoechst (blue) and F‐actin (magenta). Magnified images from boxed regions shown. Arrowheads, positive staining. Scale bars, 5 μm. Representative of n = 5 spheroids imaged. (D) Intensity profiles for active FAK and Src (grey) and F‐actin (magenta) from spheroids in (C). Tip measured is annotated, ECM to body, yellow arrow, tip, white arrowhead. E–H Representative capture ELISA graphs (E, G) and associated quantitation (F, H) for recycling of internalised cargoes between Trp53 −/− versus Trp53 −/− ; Pten −/− cells or Trp53 −/− ; Pten −/− cells expressing sh Scramble versus sh Arf6 for active β1‐integrin. Graphs shown are representative of n = 2 (E) or n = 3 (G) independent replicates. Data, mean (black square) ± SD for repeated experiments (large circles), 1–3 technical replicates/experiment/timepoint (small circles), two‐tailed t ‐test, P ‐values are annotated. I–K Overall survival (% patients, months; TCGA OV data set) of patients grouped into combined expression based on median mRNA split. (I) Low (red line, M1) or high (blue line, M2) expression for all mRNA, control, remaining patients (green line), (J), same as (I), but CYTH2 Ex9 PSI, rather than total CYTH2. (K), as for (I), but PTEN protein levels split by quantiles (red and blue, Q1 + Q2, Q3, low PTEN, green Q4, high PTEN). Median survival, sample size ( n ) and P ‐value, Log‐rank test (Mantel‐Cox) annotated. L Differential abundance ( x , Log Ratio between conditions; y , Log 10 q ‐values) of proteins in PIP 3 ‐responsive module (ARF6 HI ‐AGAP1 HI ‐CYTH2 2G ) versus PI(4,5)P 2 ‐responsive ARF module (ARF6 HI ‐AGAP1 HI ‐CYTH2 3G ) protein samples. Reverse Phase Protein Array Data, TCGA OV. Significantly altered components in AKT signalling pathway labelled (−Log 10 q ‐value > 1.3). M Schema, molecular model for ARF GTPase regulation of integrin‐dependent invasion. Source data are available online for this figure.

Article Snippet: Antibodies used were: anti‐2A peptide (Merck, MABS2005, 3H4), anti‐AKT pan (CST, 2920, 40D4), anti‐ARF5 (Novus Biologicals, H0000281‐M01, IB4), anti‐ARF6 (Merck, A5230), anti‐GAPDH (CST 2118, 14C10, 1:5,000), anti‐α5 integrin (Abcam, ab150361), anti‐β1 integrin (Merck, clone MB1.2), anti‐AKT phospho S473 (CST, 4060, D9E), anti‐S6RP phospo S235/236 (CST, 2217, 5G10), anti‐S6RP (CST, 2217, 5G10), PTEN (CST, 9552), anti‐RFP (used to detect BFP; Thermo Fisher Scientific, R10367), anti‐Streptavidin‐Horseradish Peroxidase (HRP) Conjugated (Thermo Fisher Scientific, SA10001), anti‐TP53 (Abcam, ab26, diluted in 5% milk in TBS‐T), anti‐V5 Tag (ABM, G189), anti‐Vinculin (Merck, V9131, 1:2,000).

Techniques: Immunofluorescence, Imaging, Staining, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Expressing, Two Tailed Test, Protein Array

Journal: Nature Communications

Article Title: A proteomic approach reveals integrin activation state-dependent control of microtubule cortical targeting

doi: 10.1038/ncomms7135

Figure Lengend Snippet: ( a ) Immunofluorescence microscopy revealed the morphology of HFF cells spread on stimulatory and inhibitory anti-β1 integrin mAbs compared with those spread on FN and PDL. Cells were stained for actin (red) and vinculin (green). Scale bar, 10 μm. Inset images correspond to areas highlighted in white dotted boxes. ( b ) Workflow for the isolation and proteomic analysis of integrin activation state-dependent adhesion complexes from K562 cells using paramagnetic beads coated with activation state-specific anti-β1 integrin mAbs. The mAb-coated beads recruited integrins and associated proteins in live cells, and complexes were then stabilized with crosslinker and crosslinks cleaved under reducing conditions during extraction. Proteins were then separated by SDS–PAGE, and the whole lane was cut into 30 slices, which were subjected to in-gel trypsin digestion for analysis by MS. MS data for each adhesion complex isolation were acquired in technical duplicate, from duplicate biological isolations. ( c ) The distribution of proteins identified in active and inactive integrin data sets illustrated as a Venn diagram. ( d ) Hierarchical clustering analysis of the quantitative MS data. Pearson correlation coefficients ( r ) are indicated at dendrogram nodes; a threshold of r ≥0.80 was used to identify clusters of distinct protein enrichment (red, active integrin; blue, inactive integrin; grey, unenriched). Accompanying heat bar (bottom) indicates the distribution of reported adhesome components . Bin, 20 proteins.

Article Snippet: Cells were spread on either FN, PDL or anti-β1 integrin mAbs coated on glass-bottom dishes at 10 μg ml −1 (MatTek) or on micropatterned coverslips (as described above) in serum-free DMEM containing 25 mM HEPES at 37 °C, 8% (v/v) CO 2 for 1 h. If required, cytochalasin D at a final concentration of 20 μM, nocodazole at 10 μM or equivalent volume of dimethylsulphoxide vehicle was added and incubation continued as needed by the experiment.

Techniques: Immunofluorescence, Microscopy, Staining, Isolation, Activation Assay, Extraction, SDS Page, Tandem Mass Spectroscopy, Protein Enrichment

Journal: Nature Communications

Article Title: A proteomic approach reveals integrin activation state-dependent control of microtubule cortical targeting

doi: 10.1038/ncomms7135

Figure Lengend Snippet: ( a ) The interaction network of adhesome components identified by MS was arranged according to the number of reported protein interactions (hops) from β1 integrin (ITGB1). Proteins were clustered by their detection in active (left), inactive (right) or both (middle) integrin data sets. NC, not connected. ( b ) The identified adhesome network was arranged according to functional class . Arrowheads on coloured bars indicate median protein enrichment for each class; grey bars indicate proportion of reported adhesome class identified by MS. Sphingolipid regulator quantification was derived from ‘other’ adhesome class. ‘Channel’ and ‘E3 ligase’ adhesome classes were not represented (0% identified). Nodes (proteins) are coloured according to their enrichment in active (red) or inactive (blue) integrin complexes (log 2 transformed). Gene symbols are shown for clarity (see for protein names).

Article Snippet: Cells were spread on either FN, PDL or anti-β1 integrin mAbs coated on glass-bottom dishes at 10 μg ml −1 (MatTek) or on micropatterned coverslips (as described above) in serum-free DMEM containing 25 mM HEPES at 37 °C, 8% (v/v) CO 2 for 1 h. If required, cytochalasin D at a final concentration of 20 μM, nocodazole at 10 μM or equivalent volume of dimethylsulphoxide vehicle was added and incubation continued as needed by the experiment.

Techniques: Functional Assay, Protein Enrichment, Derivative Assay, Transformation Assay

Journal: Nature Communications

Article Title: A proteomic approach reveals integrin activation state-dependent control of microtubule cortical targeting

doi: 10.1038/ncomms7135

Figure Lengend Snippet: ( a ) Overrepresented cellular component terms from proteins identified by MS were hierarchically clustered according to protein enrichment in active (red) or inactive (blue) integrin complexes. This identified clusters of similarly enriched proteins associated with a similar set of functional terms. Arrowheads indicate clusters of proteins assigned focal adhesion and microtubule-associated terms. Accompanying heat bars (right) indicate median protein enrichment (log 2 transformed) and false discovery rate-corrected P value (all <0.05; log 10 scale) for each cellular component term. Grey bars (right) highlight focal adhesion and cytoskeleton terms. ( b ) Additional annotation of the cellular component terms on the functional enrichment map in a revealed the range and specificity of cellular localizations reported for proteins enriched in active and inactive integrin complexes. Clusters containing at least eight proteins were labelled in addition to the cell adhesion terms highlighted in a . MTOC, microtubule-organizing centre; snRNP, small nuclear ribonucleoprotein.

Article Snippet: Cells were spread on either FN, PDL or anti-β1 integrin mAbs coated on glass-bottom dishes at 10 μg ml −1 (MatTek) or on micropatterned coverslips (as described above) in serum-free DMEM containing 25 mM HEPES at 37 °C, 8% (v/v) CO 2 for 1 h. If required, cytochalasin D at a final concentration of 20 μM, nocodazole at 10 μM or equivalent volume of dimethylsulphoxide vehicle was added and incubation continued as needed by the experiment.

Techniques: Protein Enrichment, Functional Assay, Transformation Assay

Journal: Nature Communications

Article Title: A proteomic approach reveals integrin activation state-dependent control of microtubule cortical targeting

doi: 10.1038/ncomms7135

Figure Lengend Snippet: Activation state-dependent recruitment of microtubule plus-end tracking proteins (+TIPs) to integrin complexes.

Article Snippet: Cells were spread on either FN, PDL or anti-β1 integrin mAbs coated on glass-bottom dishes at 10 μg ml −1 (MatTek) or on micropatterned coverslips (as described above) in serum-free DMEM containing 25 mM HEPES at 37 °C, 8% (v/v) CO 2 for 1 h. If required, cytochalasin D at a final concentration of 20 μM, nocodazole at 10 μM or equivalent volume of dimethylsulphoxide vehicle was added and incubation continued as needed by the experiment.

Techniques: Activation Assay

Journal: Nature Communications

Article Title: A proteomic approach reveals integrin activation state-dependent control of microtubule cortical targeting

doi: 10.1038/ncomms7135

Figure Lengend Snippet: ( a ) Enrichment of talin and three +TIPs, EB1, ACF7 and CKAP5, in complexes associated with active β1 integrin shown by western blotting (see for original blots). ( b ) HFFs spread on FN, stimulatory and inhibitory anti-β1 integrin mAbs stained for actin (red) and α-tubulin (green), with corresponding high-power images highlighting the difference in the location of MTs at the cell periphery in cells spread on the inhibitory mAb. MT density was calculated by counting the number of MTs within a 5 × 2 μm region of the cell periphery. Results are mean±s.d. ( n =9, 10 and 8 cells for FN, stimulatory and inhibitory, respectively). ( c ) HFFs spread on FN, stimulatory and inhibitory mAbs for 1 h before treatment with 10 μM nocodazole for 45 min and subsequent washout for a further 45 min to examine MT regrowth. Cells were stained for tubulin; dotted line in bottom-right image indicates cell periphery. MT density was measured as in b . Results are mean±s.d. ( n =3, 3 and 4 cells for FN, stimulatory and inhibitory, respectively). ( d ) HFFs spread on stimulatory and inhibitory mAbs for 1 h before addition of 20 μM cytochalasin D or dimethylsulphoxide (DMSO) vehicle control for a further 1 h. Cells were stained for actin (red) and α-tubulin (green); dotted line in bottom-right image indicates cell periphery. MT density was measured as in b . Results are mean±s.d. ( n =5 and 5 DMSO-treated cells and 5 and 7 cytochalasin D-treated cells for stimulatory and inhibitory, respectively). Scale bars, 10 μm. *** P <0.001, **** P <0.0001; one-way analysis of variance with Tukey’s post hoc correction in b , two-way analysis of variance with Tukey’s post hoc correction in c and d (see for statistics source data). Inhib., inhibitory; MW, molecular weight; NS, nonsignificant; Stim., stimulatory.

Article Snippet: Cells were spread on either FN, PDL or anti-β1 integrin mAbs coated on glass-bottom dishes at 10 μg ml −1 (MatTek) or on micropatterned coverslips (as described above) in serum-free DMEM containing 25 mM HEPES at 37 °C, 8% (v/v) CO 2 for 1 h. If required, cytochalasin D at a final concentration of 20 μM, nocodazole at 10 μM or equivalent volume of dimethylsulphoxide vehicle was added and incubation continued as needed by the experiment.

Techniques: Western Blot, Staining, Control, Inhibition, Molecular Weight

Journal:

Article Title: CD98 activation increases surface expression and clustering of ? 1 integrins in MCF-7 cells through FAK/Src- and cytoskeleton-independent mechanisms

doi: 10.3858/emm.2008.40.3.261

Figure Lengend Snippet: CD98 engagement increases the surface expression of β1 integrin. (A) Freshly cultured MCF-7 cells were dispersed and then incubated with anti-CD98 mAb UM7F8 and secondary Ab (blue line) for 1 h. As negative controls, cells were treated with mouse IgG and secondary Ab (red line). FITC-conjugated anti-CD29 mAb was used to measure the expression level of β1 integrin on the cells treated with mAbs as described above. An FITC-conjugated mouse anti-human lgG was used as the negative control (green line). (B) Cell extracts from MCF-7 cells treated as in (A) were analyzed by Western blotting using anti-β1 integrin and anti-actin mAb.

Article Snippet: R-PE-conjugated mouse anti-β 1 integrin mAb (TDM29) was from Cymbus Biotechnology (Eastleigh, Hampshire, UK).

Techniques: Expressing, Cell Culture, Incubation, Negative Control, Western Blot

Journal:

Article Title: CD98 activation increases surface expression and clustering of ? 1 integrins in MCF-7 cells through FAK/Src- and cytoskeleton-independent mechanisms

doi: 10.3858/emm.2008.40.3.261

Figure Lengend Snippet: CD98 cross-linking enhances β1 integrin clustering. (A) MCF-7 cells were incubated with anti-CD98 mAb UM7F8 with or without secondary antibody. To determine whether cross-linking of β1 integrins induces their clustering, cells were treated with anti-β1 integrin mAb 3S3 in the presence of secondary antibody as well. Next, cells treated as described in "Materials and Methods" were analyzed by confocal microscopy. Actin cytoskeleton organization is visualized by staining with phalloidin-FITC whereas β1 integrin clustering with R-PE-conjugated anti-β1 integrin mAb. Images are from a single experiment representative of more than three so performed. Scale bar, 50 µm. Original magnification, × 400. (B) Relative intensities of β1 integrin clusters in Figure 2A were measured with LSM5120 Meta NLO software. Results are values relative to the β1 integrin signal intensity level of untreated controls, designated as 1.

Article Snippet: R-PE-conjugated mouse anti-β 1 integrin mAb (TDM29) was from Cymbus Biotechnology (Eastleigh, Hampshire, UK).

Techniques: Incubation, Confocal Microscopy, Staining, Software

Journal:

Article Title: CD98 activation increases surface expression and clustering of ? 1 integrins in MCF-7 cells through FAK/Src- and cytoskeleton-independent mechanisms

doi: 10.3858/emm.2008.40.3.261

Figure Lengend Snippet: Inhibition of FAK/Src kinases with PP2 blocks CD98-induced cell adhesion, but not surface expression and clustering of β1 integrins in MCF-7 cells. (A) The effect of PP2 and/or Mn2+ on FAK phosphorylation in MCF-7 cells treated with anti-CD98 mAb was determined by immunoprecipitaion assay. MCF-7 cells were incubated with anti-CD98 mAb and PP2 (0.2 µM) or a DMSO vehicle control in the presence or absence of 0.5 µM Mn2+ for 1 h and anti-FAK rabbit polyclonal antibody (C-20) was used to immunoprecipitate FAK from extracts of MCF-7 cells. Immunoprecipitates were blotted and probed with anti-phosphotyrosine mAb (clone PY99) and anti-FAK mAb. (B) The effect of PP2 treatment on cell adhesion rate was determined as described in Materials and Methods. In addition, whether addition of 0.5 µM Mn2+ interferes with the effect of PP2 on cell adhesion was determined by the same way. Results are expressed as mean ± SE of values relative to the adhesion rate of mouse IgG-treated controls, designated as 100%. Asterisks show a significant difference from control as follows: *P < 0.05. Additional statistical comparisons are indicated by lines. (C) MCF-7 cells were incubated with anti-CD98 mAb with or without PP2 (0.2 µM) and then analyzed by flow cytometry using FITC-conjugated anti-human β1 integrin mAb. Data represent the mean ± SE of values relative to mean values of fluorescence intensity of mouse IgG-treated controls, designated as 100%. Asterisks show a significant difference from control as follows: *P < 0.05, ***P < 0.001 (D) Confocal microscopy was performed as described in Figure 2 legend to investigate the effect of PP2 (0.2 µM) on CD98-induced clustering of β1 integrins. Scale bar, 50 µm. Original magnification, × 400.

Article Snippet: R-PE-conjugated mouse anti-β 1 integrin mAb (TDM29) was from Cymbus Biotechnology (Eastleigh, Hampshire, UK).

Techniques: Inhibition, Expressing, Phospho-proteomics, Incubation, Control, Flow Cytometry, Fluorescence, Confocal Microscopy

Journal:

Article Title: CD98 activation increases surface expression and clustering of ? 1 integrins in MCF-7 cells through FAK/Src- and cytoskeleton-independent mechanisms

doi: 10.3858/emm.2008.40.3.261

Figure Lengend Snippet: The effects of dominant-negative variants of FAK on adhesiveness of MCF-7 cells, surface expression and clustering of β1 integrins. (A) MCF-7 cells were stably transfected with dominant-negative mutant FAK constructs (FRNK, Y397F-FAK) or control vector, pcDNA3. The expression levels of endogenous FAK, FRNK and Y397F-FAK were determined by Western blot analysis using anti-FAK polyclonal antibody. (B) The effect of dominant-negative variants of FAK on CD98-induced cell adhesion rate was determined as described in Materials and Methods. In addition, whether addition of 0.5 µM Mn2+ interferes with the effect of PP2 on cell adhesion was determined by the same way. Results are expressed as in Figure 3 (B). *P < 0.05, **P < 0.01 (C) FAK variants- or mock-transfected MCF-7 cells were treatred with anti-CD98 mAb and secondary antibody and then analyzed for expression of β1 integrin through flow cytometry using FITC-conjugated anti-human β1 integrin. Results are expressed as in Figure 3 (C). Statistical comparisons are indicated by lines. **P < 0.01 (D) Confocal microscopy was performed as described above to investigate the effect of dominant-negative variants of FAK on clustering of β1 integrins. Scale bar, 50 µm. Original magnification, × 400.

Article Snippet: R-PE-conjugated mouse anti-β 1 integrin mAb (TDM29) was from Cymbus Biotechnology (Eastleigh, Hampshire, UK).

Techniques: Dominant Negative Mutation, Expressing, Stable Transfection, Transfection, Construct, Control, Plasmid Preparation, Western Blot, Flow Cytometry, Confocal Microscopy

Journal:

Article Title: CD98 activation increases surface expression and clustering of ? 1 integrins in MCF-7 cells through FAK/Src- and cytoskeleton-independent mechanisms

doi: 10.3858/emm.2008.40.3.261

Figure Lengend Snippet: Cytochalasin D or phalloidin treatment inhibits CD98-induced adhesion of MCF-7 cells to fibronectin, but not surface expression and clustering of β1 integrins in MCF-7 cells. (A) MCF-7 cells were incubated with anti-CD98 mAb in the presence or absence of cytochalasin D (4 µM) or phalloidin (10 µM) for 1 h. The effects of DMSO vehicle control are also shown. The effect of cytochalasin D treatment on cell adhesion was determined as described in Materials and Methods. In addition, whether addition of 0.5 µM Mn2+ interferes with the effect of cytochalasin D or phalloidin on cell adhesion was determined by the same way. Results are expressed as Figure 3B. *P < 0.05, **P < 0.01, ***P < 0.001 (B) MCF-7 cells treated with anti-CD98 mAb in the presence or absence of cytochalasin D or phalloidin were analyzed for expression of β1 integrin using flow cytometry. Results are expressed as in Figure 3 (C). **P < 0.01 (C) Confocal microscopy was performed as described above to investigate the effect cytochalasin D or phalloidin of on clustering of β1 integrins. Scale bar, 50 µm. Original magnification, × 400.

Article Snippet: R-PE-conjugated mouse anti-β 1 integrin mAb (TDM29) was from Cymbus Biotechnology (Eastleigh, Hampshire, UK).

Techniques: Expressing, Incubation, Control, Flow Cytometry, Confocal Microscopy