E-Cadherin Expression in GBM Figure 4. E-cadherin expression in conventional glioma cell lines is rare. Nineteen conventionally grown glioma cell lines were examined by Western Blot for expression of various cadherin and catenin proteins. a-tubulin serves as a loading control. Positive control lysates are as in Figure 2. SF767 was the only conventional glioma cell line examined that expressed E-cadherin. doi:10.1371/journal.pone.0013665.g004 in peri-nuclear areas and along cell edges without adjacent cells. Even at cell-cell contact areas, many of the cells lacked tight junctional staining, and exhibited gaps and/or filopodia protru- sions (illustrated for example by the top arrow in Figure 5B). These features, combined with the lack of actin re-organization, suggested the absence of mature adherens junctions. The apparent lack of mature adherens junctions in the SF767 cells raised the question as to whether the E-cadherin expressed in these cells might be mutated. Accordingly, E-cadherin mRNA was isolated from these cells, converted to cDNA, and sequenced. No mutations in the amino acid coding sequence were found (Lewis-Tuffin, data not shown). Taken together, these observations suggest that E-cadherin-mediated, cell contact-dependent signal- ing may be deregulated in SF767 cells. To begin to address the role of E-cadherin in SF767 growth and migration, a lentiviral-based RNA knockdown approach was used. Figure 5. SF767 cells lack junctional organization of actin and have disorganized adhesive structures containing E-cadherin. A. Immunofluorescence for E-cadherin expression and actin localization was carried out on paraformaldehyde-fixed/Triton X-100-permeabilized SF767 and MCF7 cells (as a control). Arrows indicate areas of cell-cell contact; arrowheads point to areas of the plasma membrane without cell-cell contact. MCF7 cells form compact cell-cell adhesions to which the actin cytoskeleton and E-cadherin tightly localize. In contrast, E-cadherin localization in SF767 is at cell-cell contacts and on the plasma membrane at areas without cell-cell contact. Additionally, the actin cytoskeleton is not properly organized at areas of cell-cell contact. 63X magnification. The scale bar is 10 mm and applies to all images in Figure 5. B. Immunofluorescence for E- cadherin and b-catenin expression was carried out on MeOH-fixed/permeabilized SF767 and MCF7 cells. Arrows and arrowheads are as in A. b-catenin and E-cadherin both localize tightly to adherens junctions between MCF7 cells. In contrast, fewer proper cell-cell junctions exist in the SF767 cells. Both E-cadherin and b-catenin localization is diffusely distributed on the plasma membrane, in addition to a disorganized presence at areas of cell contact. 63X magnification. doi:10.1371/journal.pone.0013665.g005 PLoS ONE | www.plosone.org 5 October 2010 | Volume 5 | Issue 10 | e13665