FIGURE 9.

Decrease of endoB2 expression by siRNA interference and incidence on the vimentin network. HeLa/endoB2-St cells were transfected with control or endoB2 siRNAs (A, si endoB2-1 and si endoB2-2; B and C, si endoB2-2) for 72 h. A, Western blot analysis of endoB2 in cell lysates (top panel) or isolated after StrepTactin-Sepharose affinity chromatography (SA/seph eluate, bottom panel). In all experiments, endoB2 siRNAs allowed decreasing endoB2 expression by 75% at least in comparison with control. B, top and bottom panels correspond to two different fields. Images result from z-projections of five z-sections, each separated by a distance of 0.260 μm and obtained from cells processed for immunofluorescence using anti-vimentin antibodies. Note the mild bundling of vimentin filaments at the surface of nuclei accompanied by cytoplasmic extensions (arrowheads). Similar results were obtained with endoB2-1 siRNAs. Scale bar, 20 μm. C, top panel is a magnification of the region delimited in the top right image in B and shows the nuclear deformation imposed by the bundling of vimentin filaments. Increase in the indentation of nuclei was scored by the solidity index that measures the ratio between the surface of deformed nuclei divided by the theoretical surface whose contour is defined by the tips of indentations (see schematics). Right panel: n = 160 cells for si control and n = 153 cells for si endoB2-2.