siRNA knocking down of tetraspanins CD9 and CD151 in HUVECs. (A) Analysis by Western blot of the expression of tetraspanins CD9 and CD151 in total-cell lysates of siRNA-transfected cells. Loading control for vimentin is also shown. Densitometric analysis of the experiment shown gave a reduction in the total protein amount of 70% for CD151 and 40% for CD9 expression. (B) Flow cytometry analysis of the expression of tetraspanins (anti-CD9 VJ1/20, anti-CD151 LIA1/1, anti-CD81 I.33.2.2) in tetraspanin siRNA-treated HUVECs. Negative siRNA-treated cells are shown in thin lines. Thick lines correspond to the expression in tetraspanin siRNA-transfected cells. Negative control PX63 is shown in dotted lines. The histograms are depicted in a logarithmic scale. (C) Quantitative analysis of the mean fluorescence intensity of CD9, CD151, and CD81 in tetraspanin siRNA-transfected cells. Data represent the mean of 2 independent experiments ±SD as the percentage of the expression referred to that of negative control siRNA-transfected cells. (D) Immunofluorescence analysis of CD9 (i-iii), CD151 (iv-vi), and CD81 expression (vi-ix) and localization in HUVECs transfected with siRNA specific for endothelial tetraspanins CD9 and CD151 as well as the negative oligonucleotide. Images were acquired by confocal microscopy using the same photomultipliers parameters in control and tetraspanin-interfered cells. Maximum projection of the whole confocal image stack is shown. Scale bar equals 40 μm.