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Fig. 9. K8 knockdown and wound closure in HeLa and Panc-1 cell lines. (A) Control and K8 siRNA transfected HeLa monolayers. Red channel (left), K8 immunofluorescence, green (middle) desmoplakin immunofluorescence and merged image (right are shown). (B) Western blotting of keratin and vimentin expression in the transfected cells. HeLa cells are vimentin positive but express both keratin 8 and desmoplakin. siRNA transfection effectively knocks down K8 expression in 72 hours. (C) Panc-1 monolayer transfected with K8 siRNA and stained for K8 (red) and desmoplakin. (D) Higher magnification example of desmoplakin (green) staining at Panc 1 cell borders showing punctate desmosomal staining between K8-positive (red, left panel) but not between K8 knockdown cells (right panel). (E) Phase-contrast micrographs of scratch wound closure (20 hours time-point) in Panc-1 and HeLa monolayers transfected with control or K8 siRNA oligonucleotides. Note that the wounds in Panc-1 and HeLa monolayers were wider than in experiments with MCF-7 cells. (F) Quantification of the wound closure. Open wound at 20 hours time point was measured from photomicrographs (six measurements each from two transfections for both cell lines). Scale bar: 20 µm in A and C; 5 µm in D.
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