Specificity 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.
Linhares-Lacerda et al. BMC Genomics 2010 11(Suppl 5):S2 doi:10.1186/1471-2164-11-S5-S2