Figure 3: Ser177 phosphorylation of Pdlim5 by AMPK inhibited directional migration of vSMCs.
From: Augmented AMPK activity inhibits cell migration by phosphorylating the novel substrate Pdlim5
(a) Establishment of the KDR system for Pdlim5 in vSMCs. vSMCs were transfected with either siCTL or siPdlim5-2. siPdlim5-2-resistant EGFP-Pdlim5 (WT, S177A or S177D) was added back by adenoviral-mediated gene delivery. TCLs were subjected to immunoblotting. (b) GFP and immunostained images of KDR/EGFP-WT-Pdlim5 cells stained with a α-actinin antibody and phalloidin. Scale bar, 10 μm. (c) KDR/EGFP-Pdlim5 (WT and S177A) cells were treated with AICAR for 60 min, whereas KDR/EGFP-S177D-Pdlim5 cells were not treated with AICAR. TCLs from each cell were subjected to immunoblotting. (d) Scratch assay of KDR/EGFP-Pdlim5 cells. Phase-contrast microscopy images of KDR/Pdlim5 cells (WT, S177A and S177D) before and 8 h after scratching in the absence of AICAR. The bottom row of each panel shows analysis of migration paths over 8 h. The origins of migration of each cell were superimposed at [0, 0]. Scale bar, 0.5 mm. (e) Bar graph showing the gap width 8 h after scratching (from d). (f) Demonstration of path length (L) and displacement (D) for calculation of migration velocity and directionality. (g) Bar graph showing migration speed of each cell (from d). (h) Bar graph showing migration directionality of each cell (from d). Numbers in the bars indicate n. Data are representative of means±s.e.m. from three independent experiments. Significance of differences between series of results was assessed using one-way analysis of variance, followed by a post-hoc comparison with Dunnett’s method for multiple comparisons. *P<0.01 compared with WT.
