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Figure 8


Fig. 8. Phenotype of PKC{epsilon}-/- fibroblasts rescued by transfection with expression vector encoding constitutively active Rac1. (A) FAK phosphorylation and integrin β1 expression were analyzed as described in the Materials and Methods. Western blot analysis was used to detect phosphorylated FAK, total FAK, integrin β1 and total Rac1 in PKC{epsilon}+/+ and PKC{epsilon}-/- fibroblasts cultured on fibronectin. Transfection of constitutively active Rac (caRac), compared with empty expression vector (empty), increases phosphorylation of FAK and integrin β1 expression in PKC{epsilon}-/-, as revealed by western blot analysis. Cell extracts were prepared 24 hours post-transfection. FAK phosphorylation and integrin β1 expression were not completely impaired in the absence of PKC{epsilon}. Quantitation of three independent experiments was performed using densitometry. Average±s.d. is shown (n=3, *P<0.05). (B) Adhesion. Cells cultured on fibronectin were transfected with empty expression vector or expression vector encoding activated Rac1 (caRac). Adhesion of PKC{epsilon}+/+ (WT) and PKC{epsilon}-/- fibroblasts on fibronectin and type I collagen was monitored. Cells were detached from plates by using EDTA, and equal numbers of cells were placed into individual wells of 96-well plate. Adherent cells were detected (Absorbance 450) 45 minutes post-adhesion (average±s.d., n=3). (C) Stress fiber formation. PKC{epsilon}+/+ and PKC{epsilon}-/- fibroblasts cultured on fibronectin were fixed with paraformaldeyde and stained to detect {alpha}-SMA. Cells were counterstained with DAPI to detect nuclei (blue stain). PKC{epsilon}-/- cells transfected with caRac1 show stress fibers. (D) Migration of PKC{epsilon}+/+ (WT) and PKC{epsilon}-/- fibroblasts on fibronectin was monitored, using a scratch wound assay as described in the Materials and Methods. Photographs were taken 72 hours post-transfection, 48 hours after introduction of the scratch wound. Gap size expressed as a percentage of the original wound is shown (average±s.d.; n=3). Overexpression of Rac1 significantly increased migration of PKC{epsilon}-/- fibroblasts (Student's paired t-test, *P<0.05) compared with the appropriate control.