Figure 2From: RNA interference-mediated knockdown of CD49e (α5 integrin chain) in human thymic epithelial cells modulates the expression of multiple genes and decreases thymocyte adhesionSpecificity of CD49e gene silencing using RNA interference in cultured human thymic epithelial cells. Panel A shows cytofluorometric profiles for membrane detection of CD49e (left panels) and CD49d (α4 integrin chain, right panels) subunits of fibronectin receptors in control, scramble-siRNA and CD49e-siRNA transfected cultured human thymic epithelial cells. There is no change for CD49d expression (red line), although the expression of CD49e is clearly –down-regulated in CD49e-siRNA transfected cells (red line). The vertical black lines were inserted so that to make clearer that membrane levels of CD49e (but not CD49d) were decreased in CD49e-siRNA transfected TEC. Open peaks show the fluorescence signal generated when cells were subjected to the unrelated isotype-matched monoclonal antibody. Panel B reveals that there are no changes in the expression of CD29 (β1 integrin subunit) mRNA, as determined by real-time quantitative PCR 48 hours after transfection. Results were normalized to β-glucuronidase mRNA and are expressed relative to the mRNA levels seen in scramble-siRNA transfected cells. Data seen in this panel are representative of three independent experiments performed in triplicate. Panels C and D show the expression of fibronectin at the mRNA and protein levels respectively. Although the mean levels of mRNA (defined by qPCR) was lower in CD49e-siRNA versus scramble-siRNA transfected cultured TEC, such different was not statistically significant, and the levels of the corresponding protein, herein defined by immunohistochemistry, were similar in both groups (D). Panel D reveals immunofluorescence images for detection of fibronectin (using the anti-fibronectin 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.Back to article page