Figure 5

Effects of vimentin silencing on invasion, sphere formation and Akt phosphorylation in MDA-MB-231 persistent cells. Four days after ECP treatment, MDA-MB-231 persistent cells were seeded and cultured for 24 h before transfection with 2 sequences of siRNA targeting vimentin (VIM5, VIM13). Three days after transfection, vimentin levels were determined by Western blotting (A), or flow cytometry analysis to quantify vimentinhigh sub-population (B). Cells with fluorescence intensity ? log 103 were considered as vimentinhigh. (C) Effect of vimentin silencing on invasion of persistent cells. Three days after transfection with siRNA against vimentin, MDA-MB-231 persistent cells were seeded in the top of Boyden microchambers precoated with Matrigel. Invasive cells were counted following 24 h of culture. (D) Effect of vimentin silencing on sphere formation of persistent cells. Three days after transfection with siRNA against vimentin, MDA-MB-231 persistent cells were cultured in suspension in defined medium as described in materials and methods. The number of spheres was counted under a contrast phase microscope after 7 days of culture. (E) Western blot analysis of pS473Akt in control and persistent cells. Actin was used as loading control. (F) Western blot analysis of pan-Akt and pS473Akt in vimentin silenced persistent cells. Pan-Akt was used as loading control. Quantitative graphics correspond to 3 independent experiments and illustrations are representative of 3 independent experiments. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.00001. Unpaired Student t-test.