Overexpression of the phospho-mimicking NPM/B23 S4E mutant rescues inhibition of centriole reduplication induced by Plk2 RNAi.

<p><b>A</b> U2OS GFP-Centrin1 cells were subjected to siRNA transfections (siPlk2 or GL2 as control) using Lipofectamine2000, transfected with Flag-NPM/B23 constructs for 8 h and then subjected to a centrosome duplication assay in the presence of 1.9 µg/ml aphidicolin for 75 h. Western blot analysis demonstrated the downregulation of Plk2 by specific antibodies. loading control, α-Tubulin <b>B</b> After fixing cells from experiment in (A) with methanol indirect immunofluorescence analyses were carried out with anti-Flag antibodies. Insets show enlargements of centrioles. GFP-Centrin1, green; Flag, red; DNA, blue. Scale bar, 10 µm. <b>C</b> For statistics, U2OS GFP-Centrin1 cells from indirect immunofluorescence analyses in (B) were scored for centriole reduplication (>4 GFP-Centrin1 dots/centrioles). In case of transfections, only Flag-positive cells were analyzed. In total, 200 cells were counted each in 3 independent experiments to calculate means +/− standard deviation. unt, untreated, aph, aphidicolin.