The role of nibrin in doxorubicin-induced senescence of human Vascular Smooth Muscle Cells (VSMCs).

<p>Cells were transfected with negative siRNA (−) or <i>NBN</i> siRNA (+) and afterwards cultured for three days in the presence of doxorubicin (100 nM). <b>A.</b> Downregulation of the NBS1 protein level in VSMCs using specific siRNA (60 nM). Whole cell extracts were prepared at indicated time points after treatment with doxorubicin. Expression of the indicated proteins was estimated by Western blotting, β-actin was used as a loading control. The amount of the protein in cells transfected with <i>NBN</i> siRNA was calculated by densitometry as a fraction of that present in cells transfected with negative siRNA (1). <b>B.</b> 53BP1 staining in doxorubicin-treated control cells and cells with silenced nibrin. Representative images from one of three independent experiments. Magnification 100x. <b>C.</b> 53BP1 staining in doxorubicin treated control cells and cells with silenced nibrin. Cells with DNA damage were divided into four groups based on the number of 53BP1 foci: cells without 53BP1 foci, with one focus, with 2–5 foci, with more than 5 foci. Means from three independent experiments. <b>D.</b> SA-β-Gal activity in doxorubicin treated VSMC cells. Representative images from one of three independent experiments, magnification 100x. <b>E.</b> The percentage of SA-β-Gal positive cells (a mean ± SD) from three independent experiments. <b>F.</b> BrdU incorporation assay. Control cells and cells transfected with negative siRNA or <i>NBN</i> si RNA were cultured with BrdU for 24 h. Data presented as means ± SD from three independent experiments.