CAR Modulates E-Cadherin Dynamics in the Presence of Adenovirus Type 5
Figure 4
E-cadherin is more dynamic within junctions in the presence of Ad5eGFP.
(A) Example images from a FRAP experiment on MCF7 cells expressing E-cadherin-GFP. MCF7 were transiently transfected with E-cadherin-GFP for 24 hours before being plated on glass bottomed imaging chambers. Images were captured, analysed and exported using NIS Elements AR software. An image was taken immediately after the bleach and one 5 seconds after that, and then images were taken every 15 seconds for 5 minutes. Images shown for pre-bleach, bleach (bleached region shown in white box) and the recovery of E-cadherin-GFP monitored at 15 s, 30 s and 45 s post-bleach. (B) The half-life (τ1/2) of E-cadherin recovery after photobleaching MCF7 and FLCARMCF7 cells in the absence or presence of Ad5eGFP (MCF7+Ad5 and FLCARMCF7+Ad5 respectively). E-cadherin recovery was defined from corrected intensity data fitted to a single exponential equation and calculated using the equation t1/2 = ln 0.5/−τ. Data was pooled from >12 cells per condition over 4 independent experiments +/− SEM. Significance was determined by two-way anova. ** = p<0.001 compared to MCF7 cells no Ad5eGFP; * = p<0.05 compared to FLCARMCF7 no Ad5eGFP; * = p<0.05 compared to FLCARMCF7 with Ad5eGFP. (C) Mobile and immobile E-cadherin fractions in MCF7 and FLCARMCF7 cells in the absence or presence of Ad5eGFP (MCF7+Ad5 and FLCARMACF7+Ad5 respectively). Mobile and immobile fractions were defined as the percentage recovery at plateau (mobile) and remaining non-recovered fraction at this time (immobile fraction). Significance was determined by two-way anova. * * = P<0.001 * = P<0.05. (D) FACS analysis of cell surface E-cadherin levels on MCF7 and FLCARMCF7 cells without (black bars) or with Ad5eGFP (9000 VP/cell; white bars). MFI is shown pooled from 3 independent experiments +−/SEM. Significance was determined by a two-way anova. * = p<0.05 compared to MCF7 no Ad5eGFP levels.