Figure 3

Cell-cell contact-directed HEK293ar cell survival requires the involvement of E-cadherin. A. Increased E-cadherin expression in HEK293ar cells revealed by western blotting (n = 4-6). B. Comparison of the gray scale ratio of E-cadherin/α-tubulin between HEK293 and HEK293ar cells (n = 4-6). * p < 0.05, HEK293 versus HEK293ar. C. E-cadherin expression in HEK293ar and HEK293 cells revealed by immunofluorescence under laser scanning confocal microscope (Olympus FV1000, Tokyo, Japan; n = 4-10). Magnification: × 1600. D. Western blotting to reveal E-cadherin expression in E-cadherin RNA interference (n = 4-6). E. Comparison of the gray scale ratio of E-cadherin/α-tubulin in E-cadherin RNA interference (n = 4-6). ** p < 0.01, siRNA versus scrambled. F. The effect of E-cadherin RNA interference on the cell-cell contacts formation. Magnification: × 1000. The nuclei were counterstained with DAPI. The cells were viewed in 4-6 independent sections (at least 300 cells/section). G. Flow cytometric quantification of anoikis after E-cadherin siRNA-treatment (n = 4-6). H. The effect of E-cadherein knockdown on the cell number of HEK293ar in suspension. * p < 0.05, siRNA versus scrambled. The quantification of relative cell number was done with CyQUANT^® NF Cell Proliferation Assay Kit. This kit measures the cellular DNA content via fluorescent dye binding, which is closely proportional to cell number (n = 4-10). Each value represents the mean ± SD of triplicate determinations. Results are the representative of three similar experiments.