Immunofluorescence-Based Assay for Detection of Nuclear RAD51 Foci as a Marker of Homologous Recombination Repair in Ovarian Cancer Cells

Summary

RAD51 nuclear foci are a marker of homologous recombination (HR) activity. This manuscript describes a simple immunofluorescence method to detect these foci in cancer cells, enabling assessment of HR function and supporting preclinical investigation of responses to DNA-damaging agents.

Abstract

RAD51 is a central protein in the homologous recombination (HR) pathway and is essential for the accurate repair of DNA double-strand breaks (DSBs). Following DSB formation, DNA end resection generates single-stranded DNA substrates that facilitate the recruitment and assembly of RAD51 nucleoprotein filaments at sites of damage. This process results in the formation of discrete nuclear RAD51 foci, which serve as a widely accepted functional readout of HR activity and a surrogate marker of HR proficiency. Because defects in HR are common in several malignancies, particularly ovarian and breast cancers, assessment of RAD51 foci formation has emerged as an important approach for evaluating DNA repair capacity and predicting response to DNA-damaging therapies, including platinum compounds and poly(ADP-ribose) polymerase (PARP) inhibitors, whose efficacy is strongly influenced by HR repair status. This manuscript describes a simple, reliable, and reproducible immunofluorescence-based protocol for the detection and quantification of RAD51 nuclear foci in cultured ovarian cancer cells. The method involves induction of DNA damage by ionizing radiation (IR), followed by fixation, immunostaining with antibodies against RAD51 and γH2AX, confocal microscopy, and manual quantitative analysis of RAD51/γH2AX co-localized foci. The protocol can be applied under basal conditions or after genetic and pharmacological perturbations to determine their effects on HR function. Representative results demonstrate robust induction of RAD51 foci in HR-proficient ovarian cancer cells following DNA damage, whereas RAD51 depletion markedly reduces foci formation despite comparable levels of DSBs, confirming assay specificity. Overall, this protocol provides a robust and reproducible functional assay for assessing HR competency, with broad applications in preclinical and potentially translational cancer research.

Introduction

DNA double-strand breaks (DSBs) are among the most severe forms of DNA damage because inaccurate repair can compromise genome stability and contribute to tumor development or cell death^1,2,3. To counteract these threats, cells employ several DNA repair pathways, including homologous recombination (HR), which is considered the most accurate mechanism for repairing DSBs^2,4,5. Unlike error-prone repair pathways, HR utilizes a homologous DNA sequence as a template to restore genetic information at the damaged site. Consequently, HR predominantly functions during the S and G2 phases of the cell cycle, when sister chromatids are available to serve as repair templates^2,5. Notably, HR is essential for the accurate repair of DSBs arising from endogenous sources such as replication stress, as well as exogenous insults including ionizing radiation (IR) and DNA-damaging chemotherapeutic agents^2,5.

RAD51 is a central effector of the HR pathway and plays a critical role in homology search and strand invasion during HR-mediated repair of DSBs^2,5. Following DSB formation, DNA ends undergo resection, a process initiated by the MRE11–RAD50–NBS1 (MRN) complex and associated factors that generates 3′ single-stranded DNA overhangs. These single-stranded regions are initially coated by replication protein A (RPA), which prevents secondary structure formation and stabilizes the repair intermediate^4,5,6. HR is then initiated through the coordinated actions of BRCA1, PALB2, and BRCA2, which facilitate the recruitment and loading of RAD51 onto resected DNA, displacing RPA and promoting the formation of RAD51-containing nucleoprotein filaments^4,5,6. Once assembled, these filaments search for homologous DNA sequences within the sister chromatid and catalyze strand exchange, thereby facilitating faithful restoration of the damaged DNA^4,5,6. The accumulation of RAD51 at DSB sites appears microscopically as punctate nuclear foci that can be detected by immunofluorescence^7,8,9,10. Because formation of these foci reflects successful engagement of the HR machinery, RAD51 foci analysis has become a widely used functional approach for evaluating HR capacity and DNA repair proficiency in cells^7,8,9,10. Importantly, this assay measures the ability of cells to execute an HR response following DNA damage rather than relying solely on expression levels of HR-associated genes or proteins.

Defects in HR are frequently observed in cancer, particularly in tumors harboring alterations in BRCA1, BRCA2, RAD51 paralogs, or other genes that are involved in HR, and are especially prevalent in ovarian and breast cancers^{4,11,12,13,14}. HR deficiency has important therapeutic implications, as it confers heightened sensitivity to DNA-damaging agents such as platinum therapy and to poly(ADP-ribose) polymerase (PARP) inhibitors through the principle of synthetic lethality^{4,11,12,13,14,15}. Consequently, reliable functional assays to assess HR activity are critical for both mechanistic studies and translational research.

This protocol describes an immunofluorescence-based assay for the detection and quantification of nuclear RAD51 foci following DNA damage in ovarian cancer cells. The workflow involves induction of DNA DSBs using IR, followed by immunofluorescent detection of RAD51 and γH2AX and quantification of co-localized nuclear foci by confocal microscopy. The protocol is demonstrated using HR-proficient OVCAR-8 cells and acquired PARP inhibitor–resistant ABT^R2 cells and provides a reproducible functional approach for assessing HR activity after DSB formation. Although RAD51 foci analysis is widely used as a functional measure of HR activity, available methods often focus on specific experimental settings and frequently lack detailed guidance on integrating controlled DNA damage induction, confirmation of DNA DBS formation, assay validation controls, and standardized image-based quantification. In addition, alternative approaches for evaluating HR function, including genomic scar assays, mutational signatures, and gene or protein expression analyses, provide indirect measures of HR status and do not directly assess the cellular capacity to recruit RAD51 to sites of DNA damage. Reporter-based assays such as DR-GFP offer direct measurements of HR activity but require stable genetic engineering and are not readily applicable to many experimental models. Therefore, the workflow described here combines IR-induced DNA damage with dual RAD51/γH2AX immunofluorescence staining to simultaneously verify DSB formation and quantify RAD51 recruitment to damaged chromatin. This approach provides a practical, biologically relevant, and broadly applicable method for direct functional assessment of HR competence in cultured cancer cells, while incorporating assay controls, validation steps, and standardized foci quantification procedures.

Protocol

All materials and reagents used in this protocol are listed in the Table of Materials, including manufacturer information, catalog numbers, and antibody dilutions. An overview of the protocol is shown in Figure 1.

NOTE: The ovarian cancer cell lines OVCAR-8 and ABT^R2 are used in this protocol and have been previously described^{15,16,17,18,19,20}. OVCAR-8 cells were kindly provided by Dr. Larry Karnitz (Mayo Clinic), who originally received them from Dr. Dominic Scudiero (National Cancer Institute, NIH). The ABT^R2 cells were kindly provided by Dr. Scott Kaufmann (Mayo Clinic)²⁰. ABT^R2 is a PARPi-resistant derivative of the BRCA2.-mutated ovarian cancer cell line PEO1 that acquired resistance through restoration of BRCA2 expression. Because BRCA2 restoration re-established RAD51 loading and HR activity, ABT^R2 cells serve as a representative HR-proficient model. Cell line identity was verified based on source provenance and established characteristics reported in the literature^{15,16,17,18,19,20}. Experiments were performed using low-passage cells (passages 3–12) maintained in culture for no longer than 1 month after thawing. Cell lines were routinely tested for mycoplasma contamination every 6 months using a mycoplasma detection kit, and all cultures used in this study were confirmed to be mycoplasma-free.

CAUTION: Paraformaldehyde is toxic and should be prepared and handled in a certified chemical fume hood while wearing appropriate personal protective equipment. Human-derived cell lines should be handled in accordance with institutional biosafety procedures using appropriate containment practices. Triton X-100, mounting media, and nail polish should be handled according to the manufacturer's safety recommendations. IR should be administered only by trained personnel and in accordance with institutional radiation safety policies. Dispose of chemical, biological, and radiation-associated waste according to local institutional and regulatory requirements.

1. Cell culture and maintenance

1. Culture cells in Roswell Park Memorial Institute (RPMI)-1640 growth medium supplemented with 8% fetal bovine serum in a humidified incubator at 37 °C with 5% CO₂.
   NOTE: These culture conditions are consistent with those used in our previous studies of OVCAR-8 and ABT^R2 cells^{15,16,17,18,19}. Under these conditions, both cell lines exhibit robust growth and reproducible DNA damage response phenotypes.
2. If desired, perform perturbations (e.g., gene knockdown/knockout), following optimized experimental conditions^15,17.
   NOTE: In this protocol, siRNA-mediated knockdown of RAD51. is provided as a representative example of gene perturbation prior to immunofluorescence analysis.
   1. Prepare 5 × 10⁶ cells in 180 µL RPMI-1640 medium supplemented with 8% fetal bovine serum.
   2. Add 20 µL of 20 µM siRNA solution (2 µM siRNA per transfection) and mix gently.
      NOTE: Use the following siRNAs: Non-targeting control luciferase siRNA (siLUC): 5′-CUUACGCUGAGUACUUCGA-3′; RAD51 siRNA (siRAD51.): 5′-GGGAUUUGUGAAGCCAAA-3′. All siRNAs were purchased from Horizon Discovery.
   3. Transfer the cell suspension to a 4 mm electroporation cuvette.
   4. Electroporate cells using a BTX ECM 830 electroporator with two 10 ms pulses at 280 V.
   5. Transfer the cells to complete growth medium and incubate under standard culture conditions in a 10 cm dish for 24 h.
   6. Repeat the siRNA electroporation using the same conditions described in Steps 1.2.1–1.2.4.
   7. Incubate the cells under standard culture conditions in a 15 cm dish for an additional 48 h.
   8. Seed a portion of the cells for the immunofluorescence assay as described in Step 2. Reserve the remaining cells for RAD51 knockdown validation by immunoblotting.
   9. Validate RAD51 knockdown by immunoblotting
      1. Lyse cells in ice-cold lysis buffer containing 50 mM HEPES (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 mM NaF, 30 mM sodium pyrophosphate, 1 mM Na₃VO₄, 10 mM β-glycerophosphate, and protease inhibitor cocktail (1 tablet per 10 mL lysis buffer).
      2. Clarify the lysates by centrifugation at 18,800 × g. for 5 min at 4 °C. Transfer the supernatant to a fresh microcentrifuge tube and determine protein concentration using an appropriate protein assay.
      3. Mix an appropriate amount of protein lysate with 4× Laemmli sample buffer to achieve a final 1× concentration.
      4. Add β-mercaptoethanol to a final concentration of 20%, and heat the samples at 95 °C for 12 min prior to SDS-PAGE.
      5. Separate equal amounts of protein by SDS-PAGE and transfer proteins to an appropriate membrane for immunoblot analysis.
      6. Block the membrane in 5% milk for 30 min at room temperature with shaking.
      7. Wash the membrane three times (5 min each) with 1× tris-buffered saline with tween 20 (TBST) at room temperature with shaking.
      8. Incubate the membrane with rabbit polyclonal anti-RAD51 antibody (1:1,000 dilution) or anti-ACTIN antibody (1:1000 dilution) at 4 °C for 16–18 h with shaking.
      9. Wash the membrane with 1× TBST (5 min each) at room temperature with shaking.
      10. Incubate the membrane with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG secondary antibody (1:10,000 dilution) for 1 h at room temperature with shaking.
      11. Wash the membrane with 1× TBST (5 min each) at room temperature with shaking, and detect immunoreactive bands using an appropriate chemiluminescence detection method, and confirm RAD51 depletion relative to cells transfected with the non-targeting control siRNA.

2. Cell seeding

1. When cells reach the logarithmic growth phase (approximately 70–80% confluency), aspirate the culture medium and gently wash the cells once with sterile phosphate-buffered saline (PBS) to remove residual serum.
2. Add sufficient 0.25% trypsin–EDTA solution to cover the cell monolayer (e.g., 1–2 mL per T175 flask) and incubate at 37 °C for 2–5 min, monitoring cell detachment under a microscope.
3. Once the cells have detached, immediately neutralize the trypsin by adding an equal volume of complete culture medium containing FBS.
4. Collect the cell suspension into a 15 mL conical tube
5. Determine cell viability and concentration using trypan blue exclusion and a hemocytometer or automated cell counter prior to seeding.
6. Prepare a single-cell suspension in complete growth medium at a concentration of 4 × 10⁴ cells/mL.
7. Seed 200 µL of the cell suspension into each well of an 8-well glass chamber slide to achieve a final density of 8,000 cells per well.
8. Incubate the chamber slides for 24 h at 37 °C with 5% CO₂ to allow cell attachment.
   NOTE: The number of cells to seed may vary depending on cell type. Prepare an excess cell suspension sufficient for at least one additional well to account for pipetting variability. Seed each condition in duplicate or more to ensure reproducibility. If pharmacological modulation of HR is desired, cells should be treated after overnight attachment (12–16 h post-seeding). Seeding density should be adjusted as needed to ensure that cells reach the appropriate confluency at the time of treatment and downstream analysis, taking into account expected effects of the pharmacological agent on cell proliferation or viability. Because HR predominantly occurs during S and G2 phases of the cell cycle, maintain cultures in logarithmic growth and avoid over-confluency. For experiments involving treatments that may alter cell-cycle progression, EdU incorporation, DNA-content analysis, or other cell-cycle measurements may be performed to facilitate the interpretation of RAD51 foci results. Include untreated (–IR) and irradiated (+IR) controls in each experiment to establish basal and DNA damage-induced RAD51 foci formation, respectively. When performing siRNA-mediated perturbations, include non-targeting control siRNA (e.g., siLUC) and target-specific siRNA conditions (e.g., siRAD51.). No-primary-antibody and no-secondary-antibody controls are recommended during antibody optimization to assess nonspecific staining and background fluorescence.

3. Induction of DNA damage by IR

1. Expose cells on the chamber slides to IR at a dose of 6 Gy²¹ to induce DNA DSBs.
2. Immediately return the slides to a humidified incubator at 37 °C with 5% CO₂.
3. Incubate the cells for 6 h post-IR.
   NOTE: IR was delivered using an RS-2000 Biological Irradiator operated at 160 kV and 25.0 mA (shelf position 5). Cells were irradiated at room temperature with a single 6 Gy dose (approximately 4.8 Gy/min) and immediately returned to a 37 °C incubator with 5% CO₂ for recovery. The irradiator was routinely calibrated according to manufacturer recommendations, and all procedures were performed in accordance with institutional radiation safety guidelines. The 6 h post-IR incubation has been previously established^15,17,22. Optimal timing may vary across cell types and should be optimized for each cell line.

4. Fixation and permeabilization

1. Carefully aspirate the culture medium from the wells without disturbing the cells.
2. Fix the cells using freshly prepared 3% paraformaldehyde in 1× PBS for 12 min at room temperature.
3. Gently aspirate the fixative and wash the wells three times with 500 µL of 1× PBS per well.
4. Permeabilize cells with 0.2% Triton X-100 in 1× PBS for 12 min at room temperature.
5. Wash the wells three times with 1× PBS.
   NOTE: Perform fixation, permeabilization, and washing steps gently by adding and removing solutions along the wall of each well to minimize cell detachment. Unless otherwise indicated, use 500 µL of 1× PBS per wash.

5. Blocking

1. Prepare a blocking solution by dissolving 5% (w/v) bovine serum albumin (BSA) in 1× PBS.
2. Incubate the cells in blocking solution for 60 min at room temperature to reduce non-specific antibody binding.

6. Primary antibody incubation

1. Remove the blocking solution and wash the cells three times with 1× PBS.
2. Prepare primary antibodies at appropriate dilutions in blocking solution. Rabbit polyclonal anti-RAD51 (1:1000) and mouse monoclonal anti-phospho-Histone H2AX (γH2AX) (1:1000) antibodies are used, with γH2AX serving as a marker for DSBs^23,24.
3. Add 100 µL of diluted primary antibody solution to each well.
4. Incubate the slides at room temperature for 16–18 h in a humidified chamber to prevent evaporation.
   NOTE: The two primary antibodies can be combined in the same solution for simultaneous staining.

7. Secondary antibody incubation

1. Prepare secondary antibodies at appropriate dilutions in blocking solution. Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:350) is used to detect RAD51, and Alexa Fluor 594-conjugated goat anti-mouse IgG (1:350) is used to detect γH2AX.
2. Aspirate the primary antibody solution and wash the cells three times with 1× PBS.
3. Add 100 µL of diluted secondary antibody solution to each well.
4. Incubate slides for 1 h at room temperature in the dark.
   NOTE: The two secondary antibodies can be combined for simultaneous detection. From this section onward, perform all steps in a light-protected space to minimize photobleaching.

8. Nuclear counterstaining and mounting

1. Prepare Hoechst 33342 solution at a 1:1,000 dilution in 1× PBS to stain nuclei.
2. Aspirate the secondary antibody solution and wash cells once with 1× PBS.
3. Incubate cells with diluted Hoechst 33342 solution for 90 s at room temperature.
4. Aspirate the Hoechst 33342 solution and wash cells three times with 1× PBS.
5. Remove the 1× PBS and detach the upper chamber from the glass slides, and add about 25 µL of antifade mountant to each well.
6. Place glass coverslips over the mounted cells, ensuring even contact with the mountant.
7. Remove air bubbles gently and seal edges with nail polish.
   NOTE: Nuclear counterstaining provides a reference for nuclei, facilitating accurate localization and quantification of nuclear RAD51 and γH2AX foci.

9. Imaging and quantification of RAD51 foci at DSBs

1. Image slides immediately or store at 4 °C in a slide box in the dark until analysis.
   NOTE: To minimize potential signal degradation and photobleaching, acquire images as soon as practical, preferably within 1 week of mounting.
2. Acquire images using a confocal microscope with appropriate laser lines and filter sets: Hoechst 33342 (405 nm) for nuclei, Alexa Fluor 488 (488 nm) for RAD51, and Alexa Fluor 594 (561/594 nm) for γH2AX.
3. Perform confocal imaging using a high-magnification objective suitable for resolving subcellular structures.
4. Quantify RAD51 foci manually by counting discrete nuclear foci that co-localize with γH2AX; exclude foci not co-localized with γH2AX.
   NOTE: Validate antibody performance before quantitative analysis by confirming IR-induced RAD51 and γH2AX nuclear foci in control cells and reduced RAD51 signal following RAD51 siRNA treatment. Quantify only discrete nuclear RAD51 foci that co-localize with γH2AX in nuclei with intact morphology and clear staining patterns; exclude diffuse or non-nuclear staining, non-colocalized RAD51 signals, overlapping or multinucleated cells, mitotic cells, and apoptotic cells. Maintain identical imaging settings within each experiment and apply consistent manual scoring criteria across all conditions. Analyze ~50 nuclei per condition from multiple fields of view for each biological replicate and perform at least three independent biological replicates. Report the number of RAD51/γH2AX co-localized foci per nucleus using an appropriate statistical analysis.

10. Statistical analysis

1. Quantify RAD51/γH2AX co-localized foci from approximately 50 nuclei per condition.
2. Present data as mean ± SD from at least three independent biological replicates.
3. Perform statistical comparisons using an appropriate statistical test (e.g., an unpaired two-tailed Student's t-test for two-group comparisons or a one-way ANOVA for multiple-group comparisons)
4. Consider p. < 0.05 statistically significant.
5. Perform statistical analyses and graph generation using GraphPad Prism (version 10.6.1).

Results

IR induces RAD51 foci formation at DNA DSBs in OVCAR-8 cells
To functionally evaluate HR activity, RAD51 foci formation was examined in HR proficient OVCAR-8 ovarian cancer cells following induction of DNA DSBs by IR. To establish assay specificity and confirm that observed foci represent bona fide RAD51-dependent HR events, cells were transfected with either non-targeting control luciferase siRNA (siLUC) or RAD51-targeting siRNA (siRAD51.)¹⁵

Discussion

The immunofluorescence-based detection of nuclear RAD51 foci described here provides a reliable and functional approach to assess HR activity in cultured ovarian cancer cells. Unlike genomic or transcriptomic analyses that infer HR status indirectly, RAD51 foci formation directly reflects the cell’s ability to recruit and assemble the core HR machinery at sites of DNA DSBs. As such, this assay serves as a sensitive readout of HR competency following DNA damage and is well-suited for mechanistic studies investigatin...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10% Polyacylamide gel recipe (Lower gel buffer 20 ml,ddH2O 33.4 mL, 30% Acrylamide/Bis-acrylamide (29:1) 26.6 mL. 10% APS (fresh) 200 µL  TEMED 70 µL)	Home-made	NA	Resolving gel for SDS-PAGE
10% Polyacylamide gel recipe (Upper gel buffer 10 mL, ddH2O 23.6 mL, 30% Acrylamide/Bis-acrylamide (29:1) 6.4 mL 10% APS (fresh) 160 µL  TEMED 40 µL)	Home-made	NA	Stacking geL for SDS-PAGE
100 mL Trypsin EDTA 1X, 0.25% Trypsin/2.21 mM EDTA in Sodium Bicarbonate	Corning	25-053-C1	Used to detach adherent cells during routine passaging and prior to cell seeding.
10x Phosphate Buffered Saline (PBS)	Bio-Rad	1610780	Diluted to 1× and used for washing steps, blocking solution, antibody dilutions, fixation, and permeabilization.
10X Running buffer (6 L) Tris 181.8 g +Glycene 864.0 g+SDS 60 g	Home-made	NA	Run to proteins sample in the gel
10X Transfer (6 L) Tris 181.8 g+ Glycene 864 g	Home-made	NA	Transfter the protein from gel to membrane
10x Tris Buffered saline	Bio-Rad	1706435	buffer
30%  (w/v)Acrylamide ;0.8(w/v)/Bis-acrylamide stock solution (37.5:1)	Ultrapure EC-890	EC-890	For SDS-PAGE
ABTR2 cells	Mayo clinic	NA	acquired PARPi-resistant ovarian cancer cell lines
AKT Lysis Buffer Recipe (50 mM HEPES (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 mM NaF, 30 mM sodium pyrophosphate, 1 mM Na3VO4, 10 mM 2-glycerophosphate, 10 μg/mL leupeptin, 5 μg/mL aprotinin, 5 μg/mL pepstatin, and 20 mM microcystin-LR.	Home-made	NA	Cell lysis buffer
Ammonium Persulfate	Bio-Rad	16107000	Molecular biology as a polymerization for gels an oxidizing agent used as a free-radical initiator
Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301	Sigma-Aldrich	05-636	Primary antibody used to detect γH2AX foci. Used at 1:1000 dilution
Anti-Rad51 (Ab-1) Rabbit pAb	Sigma-Aldrich	PC130	Primary antibody used to detect RAD51 nuclear foci as a marker of homologous recombination activity. Used at 1:1000 dilution
Bovine serum albumin Protease free	GoldBio	A-420-250	Used to prepare blocking solution (5% BSA in PBS) to reduce non-specific antibody binding during immunofluorescence staining.
ChemiDoc Imaging System	Bio-Rad	12003154	Imaging system
Confocal Laser Scanning Microscope	ZEISS	Zeiss LSM 780	Immunofluorescence imaging, γH2AX foci analysis, RAD51 foci quantification, protein co-localization, and 3D image acquisition
Countess II FL Automated Cell Counter	Life Technologies	AMQAF1000	Automated Cell Counter
Cover slips	Cardinal Health	M6045-10A	Placed over mounted samples to protect stained cells and enable high-resolution imaging.
ECM 830 Square Wave Electroporation System	Hbio-BTX	45-2052	Electroporation system suitable for applications for gene delivery in mammalian cells
Fetal Bovine Serum	Corning	35-010-CV	Added to RPMI-1640 (as 8%) to support cell growth and viability.
Glycine	Thermo Fisher Scientific	A13816.0C	Buffer
Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594	Thermo Fisher Scientific	A-11032	Secondary antibody used to detect γH2AX primary antibody. Used at 1:350 dilution
Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488	Thermo Fisher Scientific	A-11034	Secondary antibody used to detect RAD51 primary antibody. Used at 1:350 dilution
GraphPad Prism	GraphPad Software	version 10.6.1	Statistical analysis, graph generation, and data visualization.
HEPES 1M	Corning	25060-C1	PH maiantance buffer in the pH 7.2-7.6 range
Hoechst 33342 solution (20 mM, 5 mL)	Thermo Fisher Scientific	62249	was used for nuclear staining
Horseradish peroxidase-conjugated anti-mouse immunoglobulin G	Cell Signaling Technology	7076S	Secondary antibody used for detection of mouse (β-actin) primary antibody in Western blotting.
Horseradish peroxidase-conjugated anti-rabbitimmunoglobulin G	Cell Signaling Technology	7074S	Secondary antibody used for detection of rabbit primary antibody (RAD51) in Western blotting.
Instant non-fat dry milk	Hyvee	G461A323	Blocking reagent
Laemmli Sample Buffer (4×)	Bio-Rad	1610747	Sample Buffer
Lonza's MycoAlert Mycoplasma Detection Assays	Lonza Bioscience	LT07-218	Used to routinely confirm that cell cultures are free of mycoplasma contamination.
Lower gel Recipe is (1 L)(Tris 181.7 g+SDS 4.0 g)PH to 8.8 w/cenc HCl	Home-made	NA	Resolving gel for SDS-PAGE
Mouse monoclonal Anti-beta Actin antibody	Abcam	ab8226	Primary antibody used to detect β-actin as a loading control in Western blot analyses.
Non-targeting control luciferase siRNA (siLUC): 5′-CUUACGCUGAGUACUUCGA-3′	Horizon Discovery	NA	working concentration 20 nmol
Nunc Lab Tek II, 8-chamber slide W/cover RS Glass slide sterile	Thermo Fisher Scientific	154534	Used for seeding and culturing cells for immunofluorescence staining and confocal imaging.
OVCAR-8 cells	National Cancer Institute, (NIH)	NA	Ovarian cancer cell lines
Paraformaldehyde 16% Aqueous Solution EM Grade	Electron microscopy sciences	15710-S	Diluted to 3% and used to fix cells prior to immunofluorescence staining.
Prolong Gold antifade Mountant	Thermo Fisher Scientific	P36930	Used to mount samples and preserve fluorescence signals during confocal imaging.
Protease inhibitor	Roche / Sigma-Aldrich	11836170001	Protease Inhibitor Cocktail
Protein Assay dye reagent concentration	Bio-Rad	5000006	Protein quantification
PVDF membrane (Immobilon-P .45 µm)	Millipore	IPVH00010	Transfer membrane
RAD51 siRNA (siRAD51): 5′-GGGAUUUGUGAAGCCAAA-3	Horizon Discovery	NA	working concentration 20 nmol
RPMI-1640 (with L-glutamine and glucose)	Corning	10-041-CV	Used as the basal growth medium for culturing ovarian cancer cell lines (OVCAR-8 and ABTR2).
Sodium Chloride (NaCl)	Fisher scientific	S271-500	Buffer
Sodium dodecyl sulfate (SDS)	Bio-Rad	1610302	Buffer
Sodium Fluoride	Sigma-Aldrich	S7920	Phosphatase inhibitor
Sodium orthovanadate	Sigma-Aldrich	S6508-50G	Phosphatase inhibitor
Sodium pyrophosphate decahydrate	Sigma-Aldrich	221368	Buffer
Sodium β-Glycerophosphate Pentahydrate	Thermo Scientific Chemicals (formerly Alfa Aesar)	L03425.22	Phosphatase inhibitor
SuperSignal™ West Pico PLUS Chemiluminescent Substrate	Thermo Fisher Scientific	34580	Detection system
TEMED 50 mL	Bio-Rad	1610801	Molecular biology as a polymerization catalyst for polyacrylamide gels with help of APS 10%
Topcoat rapid dry clear nail polish	Electron microscopy sciences	72180	Used to seal coverslip edges and prevent drying or movement of mounted samples.
Triton x-100	Bio-Rad	1610407	Used for permeabilization of fixed cells to allow antibody access to nuclear proteins.
Tween-20	Sigma-Aldrich	P1379-1L	Buffer
UltraPure EDTA	Thermo Fisher Scientific	15576028	Chelator of divalent metal cations. It is suitable for biochemistry or molecular biology applications
Upper gel Recipe is (1 L) (Tris 60.6 g+ SDS 4.0 g)PH to 6.8 w/ conc HCl	Home-made	NA	Stacking geL for SDS-PAGE
Zeiss LSM 780 laser scanning confocal microscope	Carl Zeiss, Germany	NA	Used to acquire high-resolution confocal images for visualization and quantification of RAD51 and γH2AX nuclear foci.
β-Mercaptoethanol	Bio-Rad	1610710	Reducing agent