Figure 1.

Knockdown of CD49e gene in human thymic epithelial cells by RNA interference. Panel A depicts the expression of CD49e mRNA determined by realtime quantitative PCR 48 hours after CD49e-siRNA or scramble-siRNA transfection. Results were normalized to β-glucuronidase mRNA and are expressed relative to the mRNA levels in untreated (non-transfected) cells. Data are representative of three independent experiments performed in triplicate. Bars represent the mean ± SEM; the differences between groups were validated using the Student’s t test (p < 0.009). Panel B shows flow cytometry profiles of human TEC cultures, for immunochemical detection of CD49e using a specific anti-CD49e monoclonal antibody, 72 hours post-transfection with either scramble-siRNA (gray curve) or CD49e-siRNA (white curve with black contour). The open curve with gray line on the left corresponds to the fluorescence levels elicited by an unrelated isotype matched monoclonal antibody. These plots are representative of four independent experiments. In panel C, a kinetic flow cytometry analysis reveals the transient decrease on the membrane expression of CD49e, in cultured human, TEC 48, 72, 96 or 120 hours post-transfection with CD49e-siRNA. Panel D reveals immunofluorescence images for detection of CD49e (using the anti-CD49e monoclonal antibody) in human TEC cultures, 72 hours post-transfection with scramble-siRNA or CD49e-siRNA. On the left side of the panel, an untreated TEC culture was submitted to the same immunofluorescence assay. Bars = 20 µm.