Silencing β3 integrins results in increased fibroblast-dependent cell invasion.

<p>(A) Example phase contrast images from time-lapse movies of specified cells plated on fibronectin or CDM. Graph shows quantification of migration speed from time-lapse movies of cells on specified ECM proteins. Bars are mean speed (μm/min) +/− SEM, n =  at least 110 cells over 3 independent experiments. (B) Example fluorescence confocal images of organotypic cultures either containing human dermal fibroblasts (HDF) or not. MDA MB 231 cells are represented in green (blue  =  DAPI). Scale bars are 50μm. Graph shows quantification of invasion of cells in organotypic cultures. Bars represent mean invasion index +/− SEM from at least 40 different images per cell type over 4 independent experiments. *  =  p<0.01 throughout compared to equivalent control values. (C) Analysis of metastasis of control or β1kd cells in nude mice. MDA-MB-231 cells were pre-labeled with fluorescent cell trackers and intravenously injected (5×10⁵ green and 5×10⁵ red cells together) into mice. After 48 hours the cells remaining in the vasculature were stained with mouse anti-human HLA-antibody for 5 min. Cells from one lung per mouse were isolated, stained with Alexa-647 secondary antibody and quantified based on fluorescence. Results are expressed as (mean±SEM) percentage of specified cells from all cells isolated (n = 10 mice; *, p = 0.05).</p

Fibroblast-dependent cell invasion is regulated by β1-dependent modulation of RhoA activity.

<p>(A) Example confocal images of cells plated in cell-derived matrices (CDM) and stained for phalloidin-Alexa488 (green) and (P)MLC-Alexa568 (red). Bottom panels show (P)MLC channel only. Scale bars are 10 µm. (B) Example confocal images of organotypic cultures stained with antibodies to (P)MLC (left panels). MDA MB 231 (GFP) cells are shown in right panels. Scale bars are 50 µm. (C) Example projected images of >15 confocal z-slices of control or knockdown cells expressing GFP-lifeact. Scale bars are 10 µm. (D) Quantification of protrusion area as a function of total cell area calculated from images as in (C). Bars represent mean % protrusion area per cell +/−SEM from 50 cells over 3 independent experiments. ** = p<0.01, * = p<0.005. (E) Quantification of invasion of shCon cells or β 1kd cells expressing ROCK or p190RhoGEF in organotypic assays in the absence of HDF (as in (B). Bars represent invasion index+/−SEM from 25 images over 2 independent experiments. ** = p<0.01, * = p<0.005.</p

Enhanced invasion in β1-silenced cells is regulated by attenuated FAK activity.

<p>(A) Western blot of lysates from specified cells either untreated or treated with 1 µM PF228 (FAK inhibitor) for 2 hours. Blot is probed for active (P-397) or total FAK. GAPDH serves as a loading control. Numbers below represent average active FAK levels as a % of control as quantified by densitometry from 4 independent experiments +/−SEM. (B) Western blot of lysates from shCon or β1kd cells treated with vehicle control or PF-228 at 100 nM (FAKi) and probed for P-FAK (Y-397) or total FAK. (C) Example images of shCon or β1kd cells expressing FAK FERM FRET biosensor embedded in 3D gels. Images in left panel show F-actin (phalloidin) and right panels show FRET efficiency heatmaps according to pseudocolour scale bar indicated. Graph shows quantification of >30 cells per specified condition. Bars represent mean FRET efficiency+/−SEM across 5 independent experiments. ** = p<0.01, * = p<0.005. (D) Quantification of protrusion area/cell of control or β1kd cells expressing GFP-lifeact and embedded in 3D gels. Cells were treated with DMSO or PF228 at 100 nM prior to analysis. Bars represent mean+/−SEM of 45 cells each over 2 experiments. * = p<0.01 (E) Quantification of invasion of specified cells into 3D gels treated with DMSO (vehicle control) or PF-228 at specified concentrations. Bars represent mean+/−SEM or 35 images across 3 independent experiments. ** = p<0.01, * = p<0.05.</p

β1 and β3 integrins differentially contribute to RhoA activation during invasion.

<p>(A) Z-projections of >25 confocal z-stack images of specified cells expressing GFP-lifeact embedded in 3D ECM gels. Scale bar is 10 µm. Graphs show mean cell area and % of cell area occupied by membrane protrusions quantified from reconstructed confocal z-stack images of GFP-lifeact cells as shown. At least 35 cells quantified for each, error bars are SEM. * denotes p<0.01. (B) Example images and quantification of FRET analysis of RhoA activation in each cell type. Cells cultured in 3D gels either in presence or absence of human dermal fibroblasts (HDF). Bars show mean FRET efficiency (%) +/−SEM, n =  24 for each over 3 independent experiments. (D) Quantification of RhoA activation using analysis of RhoA FRET biosensor in control cells treated with control or integrin function blocking antibodies (left graph) or integrin knockdown cells plated in 3D gels in the presence of control media or conditioned media from human dermal fibroblasts (HDF). Bars are mean FRET efficiency +/−SEM, n = 30 cells over 3 independent experiments. * = p<0.01.</p

Localisation of zyxin to adhesion complexes in ROCK1- and ROCK2-depleted keratinocytes.

<p>A: HaCaT-NSC, HaCaT-ROCK1-KD and HaCaT-ROCK2-KD keratinocytes were cultured on glass coverslips and stained with an antibody against zyxin and imaged using TIRF microscopy. Representative images from a minimum of 3 separate experiments are shown. Scale bar = 25 µm B: The mean number of zyxin positive adhesion complexes was calculated as a function of the total number of paxillin-labelled complexes and analysed using an unpaired two-way Student's T-test (**p<0.01 *p<0.05).</p

Adhesion complex formation in ROCK1- and ROCK2-depleted keratinocytes.

<p>HaCaT-NSC (A, D), HaCaT-ROCK1-KD (B, E) and HaCaT-ROCK2-KD (C, F) were cultured on glass coverslips and stained with antibodies against paxillin (A–C) or phospho-Y118 paxillin (D–F) and imaged using TIRF microscopy. Representative images from a minimum of 3 separate experiments are shown. Scale bar = 25 µm. The mean number of adhesion complexes (G) and mean area of adhesion complexes stained with anti-paxillin (H) or anti-phospho-Y118 paxillin (I) was calculated from 3 separate experiments (minimum 75 cells per experiment) and analysed using unpaired two-way Student's T-test (**p<0.01 *p<0.05).</p

Stable and transient knock-down of ROCK1 and ROCK2 expression in two different keratinocyte cell lines.

<p>A: HaCaT keratinocytes stably expressing control (NSC), ROCK1 (ROCK1 KD) or ROCK2 (ROCK2 KD) shRNA vectors or B: SCC12f keratinocytes transiently transfected with NSC, ROCK1 or ROCK2 RNAi oligos were lysed and immunoblotted with antibodies against ROCK1, ROCK2 or tubulin as a loading control.</p

Reduced FAK phosphorylation in ROCK1-depleted keratinocytes.

<p>A: HaCaT-NSC, HaCaT-ROCK1-KD or HaCaT-ROCK2-KD cells were lysed and immunoblotted to analyse phosphorylation of FAK residues serine⁷³² (FAK S732) and tyrosine⁴⁰⁷ (FAK Y407). Total FAK expression was also analysed and tubulin expression were assessed as a loading control. Densitometry was used to quantitate phosphorylation of FAK tyrosine⁴⁰⁷ (B) and serine⁷³²(C). Data shown are the mean and standard error from three separate experiments and statistical analysis was carried out using a Mann-Whitney test (*** p<0.001).</p

Adhesion complex turnover in ROCK1- and ROCK2-depleted keratinocytes.

<p>Focal adhesion size and dynamics were analysed in HaCaT-NSC, HaCaT-ROCK1-KD and HaCaT-ROCK2-KD cells transiently transfected with mRFP-paxillin. Live images of adhesion complexes were taken every 20 seconds for 20 minutes and representative images are shown (A–C). Rates of adhesion complex assembly (D) and disassembly (E) were calculated. A minimum of 12 cells per population was analysed in each experiment and the data are the mean and standard error from 3 separate experiments. Statistical analysis was performed using a Mann Whitney test (**p<0.01 *p<0.05). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031423#pone.0031423.s003" target="_blank">Videos S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031423#pone.0031423.s004" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031423#pone.0031423.s005" target="_blank">S3</a> for representative movies).</p

The UBL domain of SHARPIN mediates binding to integrin.

<p>(A) Schematic representation of SHARPIN with its functional domains and the SHARPIN fragments used in this study. (B) Pull-down experiments to determine the interaction between GFP-SHARPIN (full-length or fragments) and peptides corresponding to the cytoplasmic domain of ITGAL and ITGB2. (C) Far-Western analysis of GST-SHARPIN (full-length or fragments) binding to full-length ITGAL-ITGB2 or ITGAL-ITGB2 lacking both cytoplasmic tails. Loading controls for GST-SHARPIN (full-length or fragments) and both ITGAL-ITGB2s are shown. (D) Fluorescence polarization-based titration of GST-SHARPIN (full-length or fragments) binding to an integrin peptide corresponding to the conserved domain within the cytoplasmic tail of ITGA2. Average normalized binding curves are shown (mean ± s.e.m. ***: p<0.001).</p