Figure 5

Impact of E-cadherin downregulation on localization of hDlg and activated MEK2 at the midbody. Subconfluent Caco-2/15 cells were stained with antibodies directed against total hDlg (α-NAG, red) or against E-cadherin (green) (Panel A). Caco-2/15 cells stably infected with lentiviruses encoding for a control shRNA (shGFP) or encoding an E-cadherin-specific shRNA were lysed and proteins were analyzed by Western blotting for expression of E-cadherin and β-actin (Panel C). Caco-2/15 cells stably expressing shGFP or shE-cadherin were stained with antibodies directed against E-cadherin (red) and β-tubulin (green) (Panel B), against β-tubulin alone (green) (Panel E) or against hDlg (Santa Cruz antibody, red) and phospho-MEK (green) (Panel F). In all images, DNA (blue) was visualized by DAPI staining and scale bars are 10 μm. Midbody widths were determined at their central portion for cells that were not infected (no RNAi), or stably expressing lentiviral-coded control (shGFP) or E-cadherin specific shRNAs (Panel D). Error bars represent the standard deviation across the population of cells (number of cells for each sample is indicated on the graph); asterisks indicate that the average for E-cadherin shRNA-expressing cells is statistically different from that of both control cell populations (two-tailed Student t-test) at p ≤ 0.01.