Figure 2.

DLX4 stimulates attachment of tumor cells to mesothelial cells by inducing CD44 expression. A: Equivalent numbers of vector-control (empty vector) and +DLX4 A2780 cells that stably expressed green fluorescent protein were seeded onto confluent monolayers of normal human omental mesothelial cells cultured in 96-well plates. At 1 hour thereafter, tumor cells that were attached to mesothelial cells were viewed by fluorescence microscopy and counted in five random ×200 microscopic fields per assay. Green fluorescent protein–expressing 2008 cells transfected with empty vector, nontargeting shRNA (shControl), and DLX4 shRNAs (shDLX4-A, shDLX4-B) were assayed for attachment as described for A2780 cells. B: Representative examples of mesothelial attachment of vector-control and +DLX4 A2780 cells and of +DLX4 A2780 cells transfected with nontargeting shRNA and CD44 shRNA (shCD44-A). C: Flow cytometric analysis of cell surface staining of CD44 in vector-control and +DLX4 A2780 cells and in +DLX4 A2780 cells transfected with nontargeting shRNA and CD44 shRNAs (shCD44-A, shCD44-B). Representative examples are shown of staining and mean fluorescence intensities (MFIs). Solid gray histograms represent staining with antibody (Ab) to CD44. Dotted histograms represent staining with isotype control. D: Attachment of A2780 cells to mesothelial cells was assayed as described in A. Where indicated, A2780 cells were pre-incubated for 1 hour with neutralizing Ab to CD44 or control IgG or with no Ab before seeding onto mesothelial monolayers. Data are expressed as means ± SD for values of three independent attachment assays (A and D). ^∗∗P < 0.01 versus empty vector; ^††P < 0.01 versus +DLX4 with no Ab. Scale bar = 50 μm (B).