Figure 5

Decrease of thymocyte adhesion to human thymic epithelial cells following siRNA-induced knockdown of the CD49e gene. Panel A depicts representative microscopic fields of human TEC cultures were transfected with scramble-siRNA or CD49e-siRNA, or that remained untreated. Three days post-transfection thymocytes were led to adhere for one hour, being then fixed and were stained with Giemsa. Adhered thymocytes (seen as darkly stained spots, arrows) were more numerous in untransfected cells and scramble-siRNA transfected cells, when compared to CD49e-siRNA silenced cells. Original magnification X100. Such differences in the numbers of adhered thymocytes to TEC were quantified and data are shown in panel B, in which the adhesion degree is plotted as association index, in which values correspond to mean ± SE (see material & methods section). Differences were statistically evaluated by Student’s t-test. Data are representative of four separate experiments and were significanly different between scramble-siRNA transfected cells versus CD49e-siRNA silenced cells. *p<0.02. Panel C reveals that, the significant reduction in thymocyte adhesion comprises all CD4/CD8-defined thymocyte subsets, herein ascertained by cytofluorometry, since their proportions are similar in the three conditions. For comparison, we also included the typical CD4/CD8 profile of human thymocytes before co-culture. Lymphocyte gate was defined by side-scatter and forward scatter dot plots.