Figure 3.

Vimentin induced cell motility is dependent on PKCɛ and the phosphorylation of vimentin. (A) MCF7 cells and (B) PKCɛ null (−/−) and PKCɛRE cells were transiently cotransfected with empty vector (pCMV-Script) or wild-type (pCMV-vim) human vimentin together with a luciferase encoding vector (transfection efficiency ∼80%). The bottoms of Transwell filters were coated using 10 μg/ml BSA (random motility) or FN (haptotaxis). At 36 h post-transfection, 10⁴ cells per well were allowed to migrate for 16 h. The migrated cells were detached with trypsin, lysed and quantified using a DNA dye (see Materials and methods). An aliqout of the cells was also lysed and assayed for luciferase activity and migration was normalised to transfection efficiency. Inserted panels show vimentin expression levels before and after transient transfections with pCMV-vim in these cells. (C) Immunofluorescence staining of vimentin in PKCɛRE cells is shown. The cells were transiently transfected with wt human vimentin or vimentin(S4,6,7,8,9A). At 36 h post-transfection, the cells were plated and allowed to spread on fibronectin for 30 min, following 90 min incubation either untreated (control) or 1 μM BIM-I (BIM-I). Bar 10 μm. (D) Immunofluorescence staining of endogenous mouse vimentin (green) and ectopically expressed human vimentin (red) in PKCɛRE cells are shown. The cells were transiently transfected with wt human vimentin or vimentin(S4,6,7,8,9A). At 36 h post-transfection, the cells were plated and allowed to spread on fibronectin for 1 h. Bar 10 μm. (E) PKCɛRE cells were transiently cotransfected with empty vector (pCMV-Script), wt human vimentin (pCMV-vim), vimentin(S4,6,7,8,9A) or vimentin(S6,33,38,50,55,71,82A) together with the luciferase encoding vector. Transfected cell haptotaxis towards fibronectin was assayed as above (means±s.d., n=5, ^*P<0.05, ^†P>0.05). Inserted panels show expression levels of PKCɛ and ectopic human vimentin in these cells. (F) PKCɛ null (−/−) cells were transiently cotransfected with wt human vimentin, vimentin(S4,6,7,8,9A) or vimentin(S4,6,7,8,9D) together with the luciferase encoding vector. Transfected cell haptotaxis towards fibronectin was determined by counting the number of migrated cells and migration was normalised to transfection efficiency (means±s.d., n=5, ^*P<0.05). Inserted panel shows expression levels of ectopic human vimentin in these cells.