Figure 5.

E-cadherin knockdown disrupts glandular structures. A: Western blot analysis of glands infected with lentiviruses carrying E-cadherin (E-cad) shRNAs 1 and 2 or the empty vector (FSV). The antibody recognizes two bands corresponding to precursor and mature E-cadherin. Both forms are targeted by the specific shRNAs. Membranes were reprobed with total ERK and stained with Coomassie Blue to ensure equal protein loading. B, top left: Double E-cadherin (green) and cytokeratin (red) immunofluorescence showing down-regulation of E-cadherin expression and disruption of 3D glandular morphology in cells infected with E-cadherin shRNA. Bottom left: Immunofluorescence showing loss of GM130 location in cells infected with E-cadherin shRNA. Top right: Immunofluorescence showing formation of actin stress fibers revealed by phalloidin E-cadherin shRNA-infected cells. Bottom right: Immunofluorescence showing β-catenin cytoplasmic and nuclear translocation in shRNA-infected cells. Green fluorescent protein (GFP) micrographs were captured before fixation to avoid loss of fluorescence caused by fixation and permeabilization procedures. White scale bar = 20 μm. C, top left: Representative phase-contrast pictures of glands infected with Ientiviruses carrying PTEN shRNA (FSV-PTEN) or the empty vector (FSV). Bottom left: Western blot showing down-regulation of PTEN and increased phospho (p)-Akt, p-GSK3, and cyclinD1 expression of glands infected with lentiviruses carrying PTEN shRNA or the empty vector (FSV). Right: quantification and representative micrographs of BrdU-positive cells in FSV and PTEN shRNA-transfected cells.