Genomic regions and putative target genes associated with 5yDSS in NSGCT patients.

<p>Chr: chromosomal location of copy number alteration associated with outcome</p><p>Region: chromosomal region associated with outcome</p><p>No.: number of tumors that display the copy number alteration</p><p>Alt.: copy number alteration type (gain or loss)</p><p>p-value: p-value of the association between copy number alteration and outcome</p><p>OR: odds ratio for the event</p><p>95% C.I.: 95% confidence interval of the odds ratio</p><p>Sig. Gene: number of significantly differentially expressed genes that map to region</p><p>FDR: false discovery rate of significant genes mapping to region</p><p>Genomic regions and putative target genes associated with 5yDSS in NSGCT patients.</p

Summary table indicating the IC₅₀ value (µM) for AZD2281 (left column) and cisplatin, in absence (mid-column) or in presence (right column) of AZD2281.

<p>Summary table indicating the IC₅₀ value (µM) for AZD2281 (left column) and cisplatin, in absence (mid-column) or in presence (right column) of AZD2281.</p

ECs are defective in HR repair.

<p>A) EC cells are defective in RAD51 foci assembly. The indicated cell lines were treated with a pulse of cisplatin (3.3 µM for 6 hs) and co-stained with anti-RAD51 (red) and anti-bromodeoxyuridine (BrdU, [green]) antibodies. Harrows points to representative BrdU-positive (S-phase) cells used for RAD51 quantification (see below). B) cisplatin induces a comparable damage in U2OS and EC cell lines. The indicated cell lines were treated as in A, co-stained with γH2AX (red) and BrdU (green) antibodies, and counterstained with DAPI (blue). Harrows points to representative BrdU-positive (S-phase) cells used for γH2AX quantification. C–D) quantification of the number of RAD51 (C) and γH2AX (D) foci, before and after cisplatin treatment. Data are mean value ±s.d. of two independent experiments. In C and D a minimum of 100 nuclei were counted for each cell line. Statistical analysis was performed using a paired two-tailed Student's <i>t</i>-test (P<0.05). E) 27x-1 and Tera-1 cell lines are defective in I-SceI-induced DSB,repair, by HR. Percentage of GFP+ cells measured by flow cytometry, 48 hs upon the transfection with both DR-GFP and I-SceI expression vectors (black bars, [DR-GFP+I-SceI]). Data were normalized against the transfection efficiency measured by transfecting a (constitutive) GFP-expressing vector (white bars [NZE CAG]). Data are mean value ± s.d. of three independent experiments. Statistical analysis was performed using a paired two-tail Student’s t-test (P<0.05). For more details see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051563#pone.0051563.s003" target="_blank">Fig. S3A</a>–B.</p

EC cell lines are sensitive to AZD2281 treatment.

<p>A) Colony assay of EC cells exposed over time to increasing doses of AZD2281. The somatic cell lines MCF10A was used as positive (resistant) control. The table shows the IC₅₀ value of AZD2281 for each cell line. Data are mean value ± s.d. of three (triplicates) independent experiments. B) AZD2281 treatment induces DNA damage. FACS analysis of γH2AX-positive cells upon AZD2281 treatment. The indicated cell lines were treated over time with the IC₅₀ dosage of AZD2281 and collected 6 hs up to 72 hs after initial treatment. Data are the mean value ± s.d. of two (triplicates) independent experiments. C–F) cell cycle profile of 27x-1 and NT2D1 cell lines following the treatment with AZD2281. The indicated cell lines were treated (or left untreated) as in B, and collected ad the indicated time points for cell cycle analysis. Data are the mean value ± s.d. of three independent experiments. G–H) cell cycle distribution of γH2AX-positive cells following AZD2281 treatment. Cells were treated as in B and collected, at the indicated time points, for the staining with the γH2AX antibody. Data are the mean value ± s.d. of three independent experiments.</p

ECs are defective in ICL-induced damage repair.

<p>A) EC cell lines are sensitive to cisplatin treatment. Colony surviving assay of EC cell lines treated for 6 hs with the indicated concentrations of cisplatin. The surviving fraction was monitored by following colony formation for up to 14 days after treatment. Somatic cell lines MCF10A and U2OS were used as positive (resistant) controls. The table shows the IC₅₀ value of cisplatin for each cell line. Data are mean value ± standard deviation (s.d.) of three to five independent experiments, each done in triplicate. B) The EC cell lines sensitivity to cisplatin correlates with their inability to repair ICL-induced DSBs<b>.</b> FACS profile of γH2AX-positive cells upon cisplatin treatment. EC cell lines were treated with 3.3 µM cisplatin for 6 hs and collected at the indicated time points, from the beginning of treatment, for γH2AX staining. Data are mean value ±s.d. of three independent experiments. C) The most cisplatin-sensitive EC cell lines are eliminated by apoptosis by 48 hs after treatment. The percentage of dead cells/total, following cisplatin treatment are shown. The indicated cell lines were treated as described in B and analyzed for their apoptotic elimination by FACS (sub-G1). Data are the mean value ± s.d. of three independent experiments. D–G) cell cycle profile of 27x-1 and NT2D1 cell lines following cisplatin treatment. The indicated cell lines were treated (or left untreated) as in B, and collected at the indicated time points for cell cycle analysis. In D–E t = 0 hs indicates the cell cycle profile of the cell lines at the end of cisplatin treatment (6 hs) and it is compared to t = 6 hs in F and G. Data are the mean value ± s.d. of three independent experiments H–I) distribution of γH2AX-positive cells trough cell cycle. Cells were treated as in B, collected at the indicated time points for the staining with the γH2AX antibody, and analyzed by FACS. Time t = 0 hs indicates the cell cycle profile of γH2AX-positive cells/total at the end of cisplatin treatment (6 hs). Data are the mean value ± s.d. of three independent experiments.</p

The high sensitivity of EC cell lines to cisplatin correlates with a reduced expression of ERCC1 but not XPF.

<p>A–B) Western blotting analysis of ERCC1 and XPF expression levels in five EC cell lines. U2OS, ERCC1-deficient 165TOR, XPF-deficient (XP2YO) and XP2YO-complemented (XP-F) cell lines were used as protein expression controls. β-tubulin was used as loading control. The U2OS lane comes from the same electrophoresis gel of the other cell lines. C–D) Densitometric analysis of ERCC1 and XPF expression. Results are presented as expression level respect to U2OS cell line, and normalized against the loading control (β-tubulin). Data are mean value ±s.d. of two independent experiments.</p

In EC cell lines PARP maximal activity correlates with PARP1 protein expression.

<p>A) PARP maximal activity correlates with EC sensitivity to AZD2281. The maximal PARP activity was assayed <i>in vitro</i> by measuring the levels of PAR (poly[ADP-ribose])-polymers linked to PARP (autoribosilatyon) and histone H1 (a PARP-1 substrate), in presence of activated DNA (nuclease treated DNA). Data are mean value ± s.d. of three to four independent experiments. B) Representative image of a western blotting analysis of PARP-1 expression, in the indicated cell lines. β-tubulin was used as loading control. C) densitometric analysis of PARP1 expression in the indicated cell lines. Data are the mean value ± s.d. of five independent experiments. Results are presented as expression level respect to 27x-1 cell line, and normalized against the loading control (β-tubulin). Statistical analysis was performed using a unpaired two-tail Student’s t-test (P<0.05).</p

The reduced expression of RAD51, PTEN or BRCA1, does not account for the reduced proficiency of EC cell lines, in HR-repair.

<p>A) Representative images of western blotting analysis of RAD51 and PTEN in the indicated cell lines. The nuclear extract (n) of the somatic Hela cell line were used as positive control. β-tubulin was used as loading control. B) densitometric analysis of RAD51 expression in the indicated cell lines. Data are mean value ± s.d. of four independent experiments C) densitometric analysis of PTEN expression in the indicated cell lines. Note that the observed reduction of PTEN expression in NT2D1 cell line was not statistically significant when compared with any other EC cell line (unpaired t-test p<0.05). Data are mean value ± s.d. of two independent experiments. D) Representative images of a western blotting analysis of BRCA1 protein level in the indicated cell lines. Mouse embryonic stem cells (ES) wild type (WT) or knockout for Brca1 (<i>Brca1^−/−</i>) were used as positive and negative controls respectively. E) densitometric analysis of BRCA1 expression of the indicated cell lines. Data are mean value ±s.d. of four independent experiments. In B, C and E, results are presented as expression level respect to U2OS cell line, and normalized against the loading control (tubulin).</p

AZD2281 treatment enhances EC cell lines response to cisplatin.

<p>A) Colony assay. The indicated cell lines were treated with ½ of the IC₅₀ dose of AZD2281 for 14 days, in absence (open symbols) or in presence (filled symbols) of cisplatin. In the latter case cisplatin was given for 6 hs, in presence of AZD2281 (½ of the IC₅₀ dose). After initial treatment cisplatin was washed out and cells cultured for 14 days in presence of AZD2281. Data are mean value ±s.d. of three (triplicates) independent experiments. B) AZD2281 reduces the ability of EC cell lines to overcome cisplatin-induced damage. The indicated cell lines were either treated in continuous with the ½ of the IC₅₀ dose of AZD2281 (dashed lines) or with AZD2281 plus the IC₅₀ dose of cisplatin (non-dashed lines) as described in A) for up to 72 hs. At each indicated time point cells were harvested, and stained with the anti-γH2AX antibody for FACS analysis. Data are mean value ±s.d. of two independent experiments. C) AZD2281 enhances EC apoptotic response. The indicated cell lines were treated either with AZD2281 (½ of the IC₅₀ dose, white bar) or cispaltin (IC₅₀ dose, grey bar) or with combined therapy (black bar) as described in A, for up to 72 hs, and collected for FACS analysis of the sub-G1 fraction. Data are mean value ±s.d. of tree to four independent experiments.</p