Figure 6

Specific silencing of endogenous gelsolin and its effects on cortical F-actin, in permissive lymphocytes. (A) Western blot of endogenous gelsolin knockdown in permissive lymphocytes transfected with two siRNA-GSN oligos, either individually (siRNA1-GSN or siRNA2-GSN) or combined (siRNA(1 + 2)-GSN), normalized to the expression of total α-tubulin. Cells transfected with scrambled siRNA were used as control. One representative experiment of three is shown. (B) Flow cytometry analysis of CD4, CXCR4 and CCR5 cell-surface expression in lymphocytes transfected with scrambled siRNA, siRNA1-GSN or siRNA2-GSN. (C) Confocal analysis of F-actin and endogenous gelsolin expression in siRNA(1 + 2)-GSN-treated lymphocytes, using a mixture of fluorescent Alexa-546 oligos. Fluorescent scrambled Alexa 546 was used as a control. White asterisks indicate cells in which endogenous gelsolin expression was efficiently silenced, expressing more cortical F-actin than in their neighbouring cells in which the uptake of siRNA(1 + 2)-GSN oligos was insufficient to silence gelsolin expression. xy midsections and merged images from a representative experiment are shown. Scale bar = 5 μm. (D) Line scans show fluorescence intensity profiles of cortical F-actin in silenced, siRNA(1 + 2)-GSN-treated cell and in the not silenced neighbour cell. Images correspond to the results presented in merged images in panel B, analysed along the horizontal lines drawn in panel D. Scale bar =5 μm. Histograms show the quantification of fluorescence intensity profiles of cortical F-actin in a series of cells (12 cells per series of siRNA(1 + 2)-GSN- and scrambled-treated cells) analysed by line scanning confocal microscopy (8 cortical points analyzed per cell). (E) Flow cytometry analysis of F-actin in scrambled- (control, taken as 100% F-actin expression) or siRNA(1 + 2)-GSN-treated cells. In B and E, data are mean ± s.e.m of three independent experiments carried out in triplicate. **p < 0.01, *p < 0.05, Student’s t-test.