MARCKS knockdown induced p27^kip1 nuclear trapping is released by co-transfection with wild-type kinase interacting with stathmin (KIS).

<p><b>A.</b> Rat aortic vascular smooth muscle A7r5 cells were transfected with either non-targeting, control siRNA or MARCKS siRNA and co-transfected with DNA plasmids expressing wild-type KIS (WT KIS), or kinase-dead mutant KIS (K54R KIS). MARCKS siRNA knockdown and expression of KIS plasmids were confirmed by Western blot analysis. <b>B.</b> Co-transfection with wild-type KIS released the nuclear trapping of p27^kip1 (white arrows) in MARCKS knockdown cells, but kinase-dead KIS (K45R KIS) did not. <b>C.</b> A total of 150 cells were scored in each treatment allowing for quantitative analysis (white arrows in Fig 5B indicate examples of cells counted as with phenotype of p27^kip1nuclear accumulation). Nuclear trapping of p27^kip1 after MARCKS knockdown was released by co-transfection with wild-type KIS but not kinase dead KIS (K45R). Scale bar = 10 μm, * denotes p<0.05. <b>D.</b> Vascular smooth muscle cells were cultured at subconfluence and they remained subconfluent for the duration of the experiment. MARCKS knockdown arrested vascular smooth muscle cell proliferation. This proliferation defect was rescued by co-transfection with wide-type KIS, but not kinase-dead KIS. Proliferation was confirmed by assessing levels of proliferating cell nuclear antigen (PCNA). <b>E.</b> Compared to CAMCs, the A7r5 cells express higher constitutive levels of p27^kip1. Experiments were performed in triplicate. Statistical significance was determined by the two-tail Student’s <i>t</i>-test. * denotes <i>p</i><0.05, NS denotes not significant.