FADU analysis in L5, S3R and S4 cells treated with doxorubicin.

<p>The percentage of double-stranded DNA, was measured in all of the cell lines using the FADU method. All of the cell lines were treated with 1 and 10 µM doxorubicin and the measurements were performed 30, 60 and 90 min after treatment with this agent. Data is calculated as the percentage of control. The values are means ± SD obtained from three independent experiments. Statistical significance was estimated using the Student’s T test.</p

The induction of cellular senescence and apoptosis upon doxorubicin treatment.

<p><b>A.</b> SA-β-Gal activity. Cells were treated for 1 day with doxorubicin (L5 - 50 nM, S3R - 10 nM and S4 - 100 nM) and then cultured in doxorubicin-free medium (1+n; n- are days of culture without doxorubicin). The bars show means ± SD. The percentage of SA-β-Gal positive cells from at least three independent experiments and representative images from one of three independent experiments. <b>B.</b> Magnification 200x. <b>C.</b> Expression of protein markers of senescence (p-p53, p53, p21 and p16) analyzed by Western blotting in untreated (0) cells and on the following days after culturing in doxorubicin-free medium (1+n), β-actin was used as a loading control. <b>D.</b> The percentage of apoptotic cells after treatment with doxorubicin estimated by the AnnexinV/7-AAD flow cytometry assay. The bars show means ± SD values. Data were obtained from three independent experiments. Statistical significance was estimated using the Student’s T test.</p

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.</p

DNA content (%) in L5, S3R and S4 cells treated with different concentration of doxorubicin.

<p>The cells were treated with doxorubicin for 24 h and DNA content was measured by flow cytometry. The percentage of DNA in all phases (including subG1) of the cell cycle is shown. Data obtained after treatment with the selected concentrations of doxorubicin, chosen for the induction of senescence, is shown in bold.</p

Levels of nibrin, p70-nibrin, MRE11, ATM and BRCA1 in the DDR complex estimated by immunoprecipitation assay.

<p><b>A.</b> Level of nibrin: wild-type (p95) and the truncated form (p70) in control (C) and doxorubicin treated (D, 1 µM/1 h) S3R, S4 and VSMCs. Expression of nibrin was analyzed by immunoprecipitation using an anti-NBS1 antibody followed by Western blotting with anti-NBS1 (upper panel). Alternatively, IP using anti-MRE11 antibody was performed followed by WB with anti-NBS1 (lower panel). MRE11 was used as a loading control. The last lane (C IP) shows the negative IP control. Note that p95 is only present in VSMCs, in which there is no p70-nibrin. <b>B.</b> ATM binding to nibrin in control and doxorubicin-treated S3R and S4 cells analyzed by immunoprecipitation using anti-NBS1 antibody (upper panel) or anti-ATM antibody (lower panel). Levels of ATM and p70 were detected by WB. Loading controls were performed in both variants of IP. <b>C.</b> Expression of BRCA1 in control and dox-treated S3R and S4 cells was analyzed by immunoprecipitation using anti-ATM antibody followed by WB using an anti-BRCA1 antibody. Loading and negative IP controls were performed as above.</p

Dose-dependent influence of doxorubicin on cell cycle arrest, apoptosis and activation of the DNA damage response (DDR).

<p><b>A.</b> DNA content, analyzed by flow cytometry, 24 h after treatment with different concentrations of doxorubicin (0–250 nM). Representative histograms from one of three independent experiments. <b>B.</b> Concentration-dependent apoptosis measured 24 h after treatment with doxorubicin (0–250 nM). The percentage of apoptotic cells was estimated by the Annexin V/7-AAD flow cytometry assay in three independent experiments. The bars show means ± SD values. Data was analyzed using the CellQuest software. Statistical significance was estimated using the Student’s T test. <b>C.</b> Expression of the DDR proteins analyzed by Western blotting in control (C) and treated with doxorubicin (D) S3R, S4 and L5 cells. Whole cell extracts were prepared 24 h after cell treatment with the following cytostatic concentrations of dox: 50 nM (L5), 10 nM (S3R), 100 nM (S4) and β-actin was used as a loading control.</p

Activation of the DNA damage response pathway upon γ-irradiation.

<p>Expression of the DDR proteins analyzed by Western blotting in control (C) and exposed to radiation (IR) S3R, S4 and L5 cells. Whole cell extracts were prepared 3 h after exposure to 4 Gy of γ-radiation, β-actin was used as a loading control.</p