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. 2011 Aug 5;6(8):e23056. doi: 10.1371/journal.pone.0023056

Figure 5. Ad5eGFP modulates CAR association with β-catenin.

Figure 5

(A) (i) A mixed population of MCF7 and FLCARMCF7 cells were plated onto vitronectin-coated coverslips and incubated with Ad5eGFP at 9000 VP/cell for 3 minutes and 10 minutes before fixation. Cells were then immunostained for the .expression and localisation of E-cadherin (AB green) and β-catenin (AC; blue). Merged images are also shown (AD). Scale bars are 20 µm. Representative images are shown from 3 independent experiments with similar results. (ii) Bar charts are quantitations of E-cadherin intensity at junctions between FLCARRFP positive cells (FLCARMCF7), or those without CAR (MCF7) and calibrated on a per pixel basis to correct for any differences in junction size/area. MCF7 junction intensity values were normalised to 1 and all values for FLCARMCF7 junctions represented as a relative value to this. Values were pooled from multiple cells and images (n = >25 junctions per condition) over three independent experiments and represented as relative mean intensity. p>0.05 for E-cadherin in FLCARMCF7 vs MCF7 at all time points. (B) Fluorescence images demonstrating Ad5eGFP attachment to MCF7 and FLCARMCF7 cells. A mixed population of MCF7 and FLCARMCF7 cells were seeded onto vitronectin-coated coverslips. Cells were incubated with Ad5eGFP-Cy5 (9000 VP/cell) for 3 minutes at 37°C before fixation with 4% PFA and imaged by confocal microscopy. First two panels from left show FLCARMCF7 cells fixed and stained for CAR using RmcB (CAR-specific antibody) [1] followed by an Alexa fluor 488-conjugated secondary antibody (first panel) and RFP. Third panel shows Ad5eGFP-Cy5 attachment to cells and fourth panel from left shows merged image indicating FLCARMCF7 cells to which Ad5eGFP–Cy5 was bound. Ad5eGFP-Cy5 faint binding to MCF7 cells is also shown in the third and fourth panels. MCF7 cells in these panels are identified by lack of CAR or RFP expression. (C) (i) MCF7 and FLCARMCF7 cells were incubated with Ad5eGFP (+Ad5) at 9000 VP/cell or FK (+FK) at 160 µg/ml for 3 min, 10 min or 6 hours. Cells were then lysed and immuno-precipitated with β-catenin or control IgG followed by probing for E-cadherin. Blots were re-probed with β-catenin to control for loading and β-catenin re-probe of FK experiment is shown as an example. (ii) Whole cell lysates used for the β-catenin IP, run on a separate gel and shown for presentation. (iii) Bar charts showing mean densitometry quantification +/− SEM from four independent experiments showing relative levels of the E-cadherin/β-catenin complex for each Ad5 and FK treatments. (D) Blots as in experiments outlined in (B) re-probed for the presence of CAR. Bar chart is mean densitometry quantification +/− SEM from four independent experiments showing relative levels of CAR within the complex for Ad5 treatment. Significance was determined by a one-way anova. * = P<0.05.