Cell culture, treatments and transfections
Derivation of the A4 progression model of pre-transformed and transformed SeOvCa cells (A4-P and A4-T cells) is described earlier [7, 16]. A4-P cells (between passage numbers 15–17) and A4-T cells (between passage numbers 37–38) were used in the study and cultured stringently to avoid the risk of cross-contamination. The A4-T cells were treated with estradiol (E2; 10 nM) for 48 h and expression analysis of c-Myc, NPM1 and RAD50 was performed. The siRNA pools of negative control, NPM1, RAD50 and XRCC5 (MISSION siRNA; Sigma Aldrich Inc.) were used for generating transient knockdown cells. The A4-T cells were treated with 4 μM cisplatin (Sigma Aldrich Inc.) for 24 h, 48 h time-points and were subsequently used for expression analysis and validation studies. In siRNA transfections, 10 nmol siRNA duplexes were transfected into A4-T cells with Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s instructions, and cells were analyzed for expression validation after 48 h of transfection. To induce DNA damage and to examine H2AX-γ and NPM1 translocalization, A4-T cells 24 h after transfection were treated with 4 μM cisplatin for 24 h before immunofluorescence analysis. The 4 μM cisplatin treatment for 48 h post 24 h transfection was given to validate protein expression levels of ATM, pATR(Str428), RAD50, NPM1, XRCC5, p53, p21, CDK1, Cyclin D1, Bcl-2 and β-actin.
Semi-quantitative reverse transcription-PCR
Trizol™ reagent (Invitrogen, USA) was used to extract total RNA from cells as per manufacturer’s guidelines . Semi-quantitative reverse transcription-PCR was performed under standard conditions as described earlier  and amplified products were resolved on a 1.5% Agarose gel; β-actin was used as internal control. Gel was run and captured under gel documentation system (Syngene; Cambridge, UK).
Antibodies, immunoblotting and quantitative analysis
Immunoblotting (IB) was performed as described earlier . Primary antibodies were used at following concentrations for IB and IF (immunofluorescence) applications; anti-NPM1 (Sigma #WH0004869M1), 1:4000(IB), 1:150(IF); anti-RAD50 (Sigma #R1653), 1:1000(IB), 1:100(IF); anti-XRCC5 (Ku86, B-1) (Santa Cruz # sc-5280), 1:2000(IB), 1:100(IF); anti-c-Myc (Origene # TA100010), 1:1000; anti-Gamma H2AX (phospho Ser139) (Abcan #ab-11174), 1:2000(IB), 1:100(IF); anti-ATM (H-248) (Santa Cruz #sc-7230), 1:1000; anti-phospho-ATR(Ser428) (Cell Signaling #2853), 1:1000(IB); anti-p53 (DO-1) (Santa Cruz # sc-126), 1:1000(IB), 1:100(IF); anti-p21 (BD #556430), 1:1500; anti-Bcl-2  (Santa Cruz # sc-130308), anti-Cyclin D1 (BD #554180), 1:1000(IB); ant-Cdk-1 (Santa Cruz # sc-53219), 1:1000; anti-α-tubulin (Sigma #T5168), 1:5000(IF); ant-γ-tubulin (Sigma #5192), 1:1000(IF); anti-MAD2 (E-17) (Santa Cruz # sc-31790), 1:100(IF), and anti-BUBR1 (N-20) (Santa Cruz # sc-16193), 1:100(IF). Probing with an anti-β-actin antibody (Clone AC-15) (Sigma # A1978), 1:10,000(IB); served as a loading control. Secondary antibodies linked with horseradish peroxidase (HRP) were used as follows: anti-rabbit (1:1500), anti-mouse (1: 1500) procured from Amersham (Pharmacia Biotech, Little Chalfont, UK). Immunoblots were scanned and densitometry for quantitative analysis was performed using a Syngene Gene Genius™ Gel Documentation System (Syngene; Scientific Laboratory Supplies; #http://www.syngene.com). Protein expression values normalized with β-actin were represented as relative expression in percentage.
Cell cycle and apoptosis assay
Cell cycle analysis of transfected cells was performed with PI (Propidium-Iodide) staining using standard procedure . Sample acquisition and data analysis was performed on FACSCalibur (Becton Dickinson, San Diego, CA, http://www.bdbiosciences.com) using ModFit analytical software. Annexin V–FITC apoptosis assay was performed as described earlier  and acquisitions were made on FACSCanto II (Becton Dickinson); DiVa software (Becton Dickinson) was used for data analysis.
Immunofluorescence staining and In-situ fluorescein cell-death detection (TUNEL) assay
Control siRNA, siNPM1, siRAD50 and siXRCC5 transfected A4-T cells were grown on cover slips for 24 h followed by treatment with cisplatin and E2 for 24-48 h wherever indicated. After treatment, media was decanted and wells washed with 1X PBS buffer. Cells were fixed with 4% paraformaldehyde and were kept for 10 min on ice. Immunofluorescence staining was performed as described earlier  using NPM1, RAD50, XRCC5, H2AX-γ and p53 antibodies; Hoechst was used for nuclear staining. Images were acquired and analyzed on confocal microscope (Carl Zeiss, Jena, Germany). Quantification of intensity of p53 nuclear foci was carried by measuring the expression intensity across nuclei dimension on Leica LAS_AF analysis platform. Intensity of p53 foci was represented on y-axis, while distance or length of nuclei is shown at x-axis. TUNEL Assay was performed as described earlier . Cells with labeling solutions (Roche) were taken as negative control. Cells were washed thrice and stained with Hoechst for nuclear staining. Stained samples were acquired and analyzed on confocal microscope (Leica, Germany).
Control, NPM1, RAD50 and XRCC5 siRNA transfected A4-T cells were grown in 24 well plate and treated with cisplatin (4 μM) and E2 (10 nM) for next 48 h. Upon harvesting, cells were washed twice with chilled 1X phosphate buffer saline (PBS) and then fixed with ice-cold absolute methanol for 20 min at 4°C. Cells were rinsed once with PBS and incubated with Giemsa stain for 30 min at RT. Further, cells were washed twice with 1X PBS and analyzed under Olympus microscopy (Olympus Co., Tokyo, Japan) at 40X magnification.
All experiments were carried out in triplicate; data are expressed as mean ± SEM of three independent experiments. The significance of difference in the mean values was determined using two-tailed Student's t test; wherein p < 0.05 considered significant. ANOVA test was performed to compare protein expression between the groups at a significance level of < 0.05. Student-Bonferroni test was used to evaluate sub-comparisons to error rate.