BRCA2 BRC missense variants disrupt RAD51-dependent DNA repair
- Department of Therapeutic Radiology, Yale University, United States
- Department of Medical Oncology, Yale University School of Medicine, Yale Cancer Center, United States
- Department of Biochemistry and Molecular Pharmacology, New York University, United States
Figures
Figure 1 with 1 supplement
BRCA2 BRC residues S1221, T1346, and T1980 are conserved residues.
S1221P and T1980I are structurally predicted to disrupt BRC folding and RAD51 binding. (A) BRCA2 protein schematic depicting domain organization: 2XMBP tag, N-terminus, BRC repeats, DNA binding …
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Figure 1—source data 1
Contains BRCA2 sequence alignment depicting homology (Figure 1B), SWISS-MODEL homology modeling reports (Figure 1C), close up views of BRC-RAD51 amino acid interactions (Figure 1C).
- https://cdn.elifesciences.org/articles/79183/elife-79183-fig1-data1-v3.zip
Figure 1—figure supplement 1
Polar contacts of S1221P, T1526A, and T1980I.
Homology model comparisons of BRC2 and BRC7 based on the BRC4 (1N0W) structure. Top Inset: Polar contacts (black dotted lines) within a BRC4 loop containing residue T1526. Bottom insets: Loss of …
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Figure 1—figure supplement 1—source data 1
T1526 polar contacts.
T1526A polar contacts. T1526I polar contacts. T1526P polar contacts.
- https://cdn.elifesciences.org/articles/79183/elife-79183-fig1-figsupp1-data1-v3.zip
Figure 2 with 2 supplements
S1221P and T1980I only partially complement BRCA2 knockout cells in response to DNA damage.
T1346I fully rescues DNA damage sensitivity and RAD51 foci formation upon irradiation. (A) Western blot of total cellular lysates from DLD-1 BRCA2-/- cells stably transfected with either empty …
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Figure 2—source data 1
Contains original gel images depicting cropped regions used for Figure 2A, clonogenic survival data for Figure 2B, foci data for Figure 2C.
- https://cdn.elifesciences.org/articles/79183/elife-79183-fig2-data1-v3.zip
Figure 2—figure supplement 1
Expression and nuclear localization of S1221P, T1346I, and T1980I BRCA2 proteins.
(A) Western blot of total cellular lysates from DLD1 and DLD-1 BRCA2-/- cells stably transfected with either empty vector (BRCA2-/-) or BRCA2 Wild Type (WT), BRCA2 S1221P (BRC2), T1346I (Spacer …
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Figure 2—figure supplement 1—source data 1
Raw Nt Ct BRCA2 RAD51.
Raw Stain Free. Nt Ct BRCA2 RAD51 highlighedbandsandlabels.
- https://cdn.elifesciences.org/articles/79183/elife-79183-fig2-figsupp1-data1-v3.zip
Figure 2—figure supplement 2
BRCA2-/- cells stably expressing full-length BRCA2 S1221P and T1980I proteins only partially rescue sensitivity to crosslinking agents (MMC or cisplatin) and PARP inhibitors (Olaparib or Talazoparib).
The T1346I protein rescues to the same extent as WT. (A) Representative images of cell colonies stained with crystal violet 14 days after treatment. PE; plating efficiency. Treatments: 2.5 µM MMC 1 …
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Figure 2—figure supplement 2—source data 1
Revised plating efficiency.xlsx.
- https://cdn.elifesciences.org/articles/79183/elife-79183-fig2-figsupp2-data1-v3.xlsx
Figure 3
S1221P and T1980I exhibit defects in HDR and fork protection.
(A) Schematic of the Clover-LMNA HDR assay. (B) Immunofluorescence images and quantification of Clover-LMNA HDR (green) and DAPI staining to visualize nuclei (blue) 96 hours post-transfection …
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Figure 3—source data 1
Contains quantification of clover positive cells (Figure 3C), images of DNA fibers (Figure 3E), DNA fiber analysis (Figure 3F).
- https://cdn.elifesciences.org/articles/79183/elife-79183-fig3-data1-v3.zip
Figure 4 with 2 supplements
BRC2-S1221P and BRC7-T1980I abolish RAD51 binding.
(A) Schematic of amylose pull-down reaction to detect RAD51 binding. (B) Total cellular lysates (TCL) and amylose pull-downs from HEK 293T cells transiently transfected with 2XMBP, 2XMBP-BRC2, …
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Figure 4—source data 1
Contains original (uncropped) gel images for Figure 4B, D, E.
- https://cdn.elifesciences.org/articles/79183/elife-79183-fig4-data1-v3.zip
Figure 4—figure supplement 1
S1221P, T1346I, and T1980I full-length BRCA2 proteins bind RAD51.
(A) Western blots of total cellular lysates (TCL) (left gel) or amylose pull-downs (right gel) from BRCA2-/- cells stably expressing full-length BRCA2 WT, S1221P, T1346I, and T1980I. 2XMBP-BRCA2 …
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Figure 4—figure supplement 1—source data 1
Contains band quantification and original (uncropped) gel images.
- https://cdn.elifesciences.org/articles/79183/elife-79183-fig4-figsupp1-data1-v3.zip
Figure 4—figure supplement 2
BRC1-2, BRC7-8, BRC2, and BRC7 bind RAD51 whereas BRC2-S1221P and BRC7-T1980I ablate RAD51 binding.
(A) Western blots of total cellular lysates (TCL) and amylose pull-downs from HEK 293T cells transiently transfected with 2XMBP or 2XMBP-BRC repeat constructs: BRC1-8, BRC1-4, BRC5-8, BRC1-2, …
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Figure 4—figure supplement 2—source data 1
Contains original (uncropped) gel images.
- https://cdn.elifesciences.org/articles/79183/elife-79183-fig4-figsupp2-data1-v3.zip
Figure 5 with 3 supplements
BRC2-S1221P and BRC7-T1980I fail to stimulate RAD51-ssDNA complex formation.
(A) Schematic of reaction to assay BRC repeat stimulation of RAD51-ssDNA complex formation by EMSA. RAD51 was pre-incubated with increasing amounts of BRC protein for 15 min then radiolabeled ssDNA …
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Figure 5—source data 1
Contains uncropped gel images (Figure 5B), EMSA band quantification (Figure 5C), biotin-DNA pulldown band quantification (Figure 5E), histogram data (Figure 5F).
- https://cdn.elifesciences.org/articles/79183/elife-79183-fig5-data1-v3.zip
Figure 5—figure supplement 1
Purified proteins used for biochemical studies.
Optimization of RAD51-ssDNA EMSA. (A) Coomassie stained gel of 2XMBP-BRC fragments BRC2, BRC2-S1221P, BRC7, and BRC7-T1980I proteins purified from HEK 293T cells. Human RAD51 and RPA proteins …
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Figure 5—figure supplement 1—source data 1
Contains original (uncropped) gel images.
- https://cdn.elifesciences.org/articles/79183/elife-79183-fig5-figsupp1-data1-v3.zip
Figure 5—figure supplement 2
smFRET control assays highlighting BRC wildtype and mutant proteins (S1221P and T1980I) show no change in FRET efficiency in the absence of RAD51.
(A) Histograms depicting: DNA only, BRC storage buffer only, individual BRC repeat proteins in the absence of RAD51 protein, and 400 nM RAD51 alone. Representative histograms do not include zero …
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Figure 5—figure supplement 2—source data 1
Contains data and traces for single molecule FRET.
- https://cdn.elifesciences.org/articles/79183/elife-79183-fig5-figsupp2-data1-v3.zip
Figure 5—figure supplement 3
Representative trajectories corresponding to histograms in Figure 5F.
Representative individual smFRET trajectories for DNA only, 20 nM RAD51, 20 nM RAD51 in presence of 20 nM BRC2 (WT or S1221P), and 20 nM RAD51 in presence of 20 nM BRC7 (WT or T1980I). The donor …
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Figure 5—figure supplement 3—source data 1
Contains data and traces for single molecule FRET.
- https://cdn.elifesciences.org/articles/79183/elife-79183-fig5-figsupp3-data1-v3.zip
Figure 6 with 1 supplement
RAD51-mediated DNA strand exchange activity is stimulated by increasing amounts of WT BRCA2 but not T1980I.
(A) Diagram of DNA strand exchange assay. RPA was pre-bound to the 3’ tail DNA substrate for 5 min. Increasing amounts of WT BRCA2 or T1980I protein was then added in combination with RAD51 for 5 …
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Figure 6—source data 1
Contains original (uncropped) gel image (Figure 6B) and band quantitation (Figure 6C).
- https://cdn.elifesciences.org/articles/79183/elife-79183-fig6-data1-v3.zip
Figure 6—figure supplement 1
Purified full-length BRCA2 WT and T1980I proteins bind a panel of DNA substrates with equivalent efficiencies.
(A) Stain Free gel (4–15% gradient SDS-PAGE) of full-length 2XMBP-BRCA2 proteins purified from 293T cells. (B) EMSAs depicting increasing concentrations of BRCA2 WT and T1980I proteins incubated …
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Figure 6—figure supplement 1—source data 1
Contains original (uncropped) gel images and band quantitation.
- https://cdn.elifesciences.org/articles/79183/elife-79183-fig6-figsupp1-data1-v3.zip
Figure 7
BRCA2 missense variants S1221P (BRC2) and T1980I (BRC7), but not T1346I (spacer BRC2-3).
(1) exhibit decrease in HDR and fork degradation, (2) disrupt RAD51 binding and (3) fail to stimulate RAD51-ssDNA complex formation and RAD51 filament stability.
Tables
Table 1
Amino acid sequences of synthesized peptides.
| PEPTIDES | AMINO ACID SEQUENCES |
|---|---|
| BRC2 | NEVGFRGFYSAHGTKLNVSTEALQKAVKLFSDIEN |
| BRC2-S1221P | NEVGFRGFYPAHGTKLNVSTEALQKAVKLFSDIEN |
| BRC7 | SANTCGIFSTASGKSVQVSDASLQNARQVFSEIED |
| BRC7-T1980I | SANTCGIFSIASGKSVQVSDASLQNARQVFSEIED |
Table 2
Amino acid sequences of BRC purified proteins.
| CONSTRUCTS | AMINO ACID SEQUENCE |
|---|---|
| 2xMBP BRC2 | YLTDENEVGFRGFYSAHGTKLNVSTEALQKAVKLFSDIENISEETSAEVHPISL* |
| 2xMBP BRC2-S1221P | YLTDENEVGFRGFYPAHGTKLNVSTEALQKAVKLFSDIENISEETSAEVHPISL* |
| 2xMBP BRC4 | RDEKIKEPTLLGFHTASGKKVKIAKESLDKVKNLFDEKEQGTSEI* |
| 2xMBP BRC4-T1526A | RDEKIKEPTLLGFHAASGKKVKIAKESLDKVKNLFDEKEQGTSEI* |
| 2xMBP BRC7 | GKLHKSVSSANTCGIFSTASGKSVQVSDASLQNARQVFSEIEDSTKQ* |
| 2xMBP BRC7-T1980I | GKLHKSVSSANTCGIFSIASGKSVQVSDASLQNARQVFSEIEDSTKQ* |
Table 3
Oligonucleotide sequences.
| Oligonucleotides | Sequence |
|---|---|
| RJ-167-mer | 5′-CTG CTT TAT CAA GAT AAT TTT TCG ACT CAT CAG AAA TAT CCG TTT CCT ATA TTT ATT CCT ATT ATG TTT TAT TCA TTT ACT TAT TCT TTA TGT TCA TTT TTT ATA TCC TTT ACT TTA TTT TCT CTG TTT ATT CAT TTA CTT ATT TTG TAT TA TCC TTA TCT TAT TTA-3′ |
| RJ-5’Tail-167mer | 5’-ATT TAT TCT ATT CCT CTT TAT TTT CTC TGT TTA TTC ATT TAC TTA TTT TGT ATT AAT TTC CTA TAT TTT TTA CTT GTA TTT CTT ATT CAT TTA CTT ATT TTG TAT TAT CCT TAT TTA TAT CCT TTC TGC TTT ATC AAG ATA ATT TTT CGA CTC ATC AGA AAT ATC CG-3’ |
| RJ-167-mer complementary | 5’-TAA ATA AGA TAA GGA TAA TAC AAA ATA AGT AAA TGA ATA AAC AGA GAA AAT AAA GTA AAG GAT ATA AAA AAT GAA CAT AAA GAA TAA GTA AAT GAA TAA AAC ATA ATA GGA ATA AAT ATA GGA AAC GGA TAT TTC TGA TGA GTC GAA AAA TTA TCT TGA TAA AGC AG-3’ |
| RJ-PHIX-42–1 | 5′-CGG ATA TTT CTG ATG AGT CGA AAA ATT ATC TTG ATA AAG CAG-3′ |
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens, Drosophila melanogaster, Mus musculus Felis catus, Rattus norvegicus, Strongylocentrotus purpuratus, Monodelphis domestica, Canis lupus familiaris, Xenopus tropicalis) | BRCA2 | Uniprot | P51587 Homo sapiens (Human); Q9W157 Drosophila melanogaster (Fruit fly); P97929Mus musculus (Mouse); Q864S8Felis catus (Cat); O35923Rattus norvegicus (Rat); A5A3F7 Strongylocentrotus purpuratus (Purple sea urchin); A4ZZ89Monodelphis domestica (Gray short-tailed opossum); Q8MKI9Canis lupus familiaris (Dog) (Canis familiaris); A4ZZ90Xenopus tropicalis (Western clawed frog) (Silurana tropicalis). | Alignment Figure 1 |
| Strain, strain background (Escherichia coli) | Stellar Competent Cells | Takara-Clontech | Cat# 636766 | Competent cells |
| Strain, strain background (Escherichia coli) | Rosetta(DE3)pLysS Competent Cells | Sigma-Aldrich | Cat# 70956 | |
| Strain, strain background (Escherichia coli) | E. coli Acella(DE3)/pCH1–RAD51o | Subramanyam and Spies, 2018 | ||
| Cell line (Homo-sapiens) | DLD1 | Horizon Discovery Hucl et al., 2008 | ||
| Cell line (Homo-sapiens) | DLD1 BRCA2 -/- | Horizon Discovery Hucl et al., 2008 | ||
| Cell line (Homo-sapiens) | HEK2093T | Jensen et al., 2010 | ||
| Recombinant DNA reagent | pBluescript BRCA2 (1–5286 bp) | This paper | Dr. Ryan Jensen laboratory. Material & Methods section | |
| Recombinant DNA reagent | pUC57 BRCA2 (2141–9117 bp) Genescript | Synthetized by Genescript | ||
| Recombinant DNA reagent | pX330-LMNA-gRNA1 | addgene | 122507 | sgRNA plasmid targeting Lamin A |
| Recombinant DNA reagent | pCR2.1 Clover-LMNA Donor | Addgene | 122507 | Donor |
| Transfected construct (Homo-sapiens) | 2XMBP-BRC2 | This paper | YLTDENEVGFRGFYSAHGTKLNV STEALQKAVKLFSDIENISE ETSAEVHPISL* | Dr. Ryan Jensen laboratory. Material & Methods section |
| Transfected construct (Homo-sapiens) | 2XMBP-BRC2-S1221P | This paper | YLTDENEVGFRGFYPAHGTKLN VSTEALQKAVKLFSDIENISE ETSAEVHPISL* | Dr. Ryan Jensen laboratory. Material & Methods section |
| Transfected construct (Homo-sapiens) | 2XMBP-BRC7 | This paper | GKLHKSVSSANTCGIFSTASGKS VQVSDASLQNARQVFSEIEDSTKQ* | Dr. Ryan Jensen laboratory. Material & Methods section |
| Transfected construct (Homo-sapiens) | 2XMBP-BRC7-T1980I | This paper | GKLHKSVSSANTCGIFSIASGKSV QVSDASLQNARQVFSEIEDSTKQ* | Dr. Ryan Jensen laboratory. Material & Methods section |
| Transfected construct (Homo-sapiens) | 2XMBP BRCA2 WT | Jensen et al., 2010 | Dr. Ryan Jensen laboratory. Material & Methods section | |
| Transfected construct (Homo-sapiens) | 2XMBP BRCA2 S1221P | This paper | Dr. Ryan Jensen laboratory. Material & Methods section | |
| Transfected construct (Homo-sapiens) | 2XMBP BRCA2 T1346I | This paper | Dr. Ryan Jensen laboratory. Material & Methods section | |
| Transfected construct (Homo-sapiens) | 2XMBP BRCA2 T1980I | This paper | Dr. Ryan Jensen laboratory. Material & Methods section | |
| Peptide, recombinant protein | RPA | This paper, Anand et al., 2018; | Dr. Ryan Jensen laboratory. Material & Methods section | |
| Peptide, recombinant protein | RAD51 | This paper, Subramanyam and Spies, 2018 | Dr. Ryan Jensen laboratory. Material & Methods section | |
| Peptide, recombinant protein | BRC2 peptide | Pierce, This paper | NEVGFRGFYSAHGTKLN VSTEALQKAVKLFSDIEN | Dr. Ryan Jensen laboratory. Material & Methods section |
| Peptide, recombinant protein | BRC2-S1221P peptide | Pierce, This paper | NEVGFRGFYPAHGTKL NVSTEALQKAVKLFSDIEN | Dr. Ryan Jensen laboratory. Material & Methods section |
| Peptide, recombinant protein | BRC7- peptide | Pierce, This paper | SANTCGIFSTASGKSVQ VSDASLQNARQVFSEIED | Dr. Ryan Jensen laboratory. Material & Methods section |
| Peptide, recombinant protein | BRC7- T1980I peptide | Pierce, This paper | SANTCGIFSIASGKSVQV SDASLQNARQVFSEIED | Dr. Ryan Jensen laboratory. Material & Methods section |
| Antibody | Anti-MBP (mouse monoclonal) | NEB | Cat# E8032L | IF (1:100) WB (1:1000) |
| Antibody | Anti-BRCA2 (rabbit polyclonal) | Abcam | Cat# ab27976 | IF (1:100) WB (1:1000) |
| Antibody | Anti-BRCA2 Nt (rabbit polyclonal) | Bethyl Laboratories | Cat# A303-434 | WB (1:1000) |
| Antibody | Anti-RAD51 (rabbit polyclonal) | Proteintech | Cat# 14961–1-AP, RRID:AB_2177083 | IF (1:100) WB (1:1000) |
| Antibody | Anti-RAD51 (mouse monoclonal) | Novus Biologicals | Cat# NB 100–148, RRID:AB_350083 | WB (1:1000) |
| Antibody | Rabbit Anti-RAD51 (rabbit polyclonal) | Abcam | Cat# ab63801, RRID:AB_1142428 | IF (1:100) |
| Antibody | Anti-BrdU (purified mouse monoclonal) | BD Biosciences | Cat# 347580, RRID:AB_10015219 | 1:10 |
| Antibody | Anti-BrdU (rat monoclonal) | Abcam | Cat# ab6326, RRID:AB_2313786 | 1:40 |
| Antibody | Anti-Rat Cy5 (goat polyclonal) | Abcam | Cat# ab6565, RRID:AB_955063 | 1:100 |
| Antibody | Anti-Mouse Cy3 (sheep polyclonal) | Sigma Aldrich | Cat# C2181, RRID:CVCL_K942 | 1:100 |
| Antibody | m-IgGκ BP-HRP | Santa Cruz Biotechnology | Cat# sc-516102, RRID:AB_2687626 | WB (1:10000) |
| Antibody | anti-rabbit IgG-HRP (goat polyclonal) | Santa Cruz Biotechnology | Cat# sc-2004, RRID:AB_631746 | WB (1:10000) |
| Antibody | anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (goat polyclonal) | Thermo Fisher Scientific | Cat# A-11034, RRID:AB_2576217 | IF (1:10000) |
| Antibody | anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 (goat polyclonal) | Thermo Fisher Scientific | Cat# A-11003, RRID:AB_2534071 | IF (1:10000) |
| Sequenced- based reagent | dT40 | Carreira and Kowalczykowski, 2011 | EMSA | 5’TTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTT3’ |
| Sequenced- based reagent | RJ-167-mer | Jensen et al., 2010 | EMSA DNA strand exchange | 5′-CTG CTT TAT CAA GAT AAT TTT TCG ACT CAT CAG AAA TAT CCG TTT CCT ATA TTT ATT CCT ATT ATG TTT TAT TCA TTT ACT TAT TCT TTA TGT TCA TTT TTT ATA TCC TTT ACT TTA TTT TCT CTG TTT ATT CAT TTA CTT ATT TTG TAT TA TCC TTA TCT TAT TTA-3′ |
| Sequenced- based reagent | RJ-5’tail 167-mer | Jensen et al., 2010 | EMSA | : 5’-ATT TAT TCT ATT CCT CTT TAT TTT CTC TGT TTA TTC ATT TAC TTA TTT TGT ATT AAT TTC CTA TAT TTT TTA CTT GTA TTT CTT ATT CAT TTA CTT ATT TTG TAT TAT CCT TAT TTA TAT CCT TTC TGC TTT ATC AAG ATA ATT TTT CGA CTC ATC AGA AAT ATC CG-3’ |
| Sequenced- based reagent | RJ-PhiX-42–1 | Jensen et al., 2010 | EMSA DNA Strand Exchange | 5’-CGG ATA TTT CTG ATG AGT CGA AAA ATT ATC TTG ATA AAG CAG-3' |
| Sequenced- based reagent | PS4 | Jensen et al., 2010 | EMSA DNA Strand Exchange | 5’- TAATACAAAATAAGTAAATGA ATAAACAGAGAAAATAAAG –3' |
| Sequenced- based reagent | PS5 | Jensen et al., 2010 | EMSA DNA Strand Exchange | 5’- CTTTATTTTCTCTGTTTATTCATT TACTTATTTTGTATTA –3' |
| Chemical compound, drug | Jet Optimus | Polyplus Transfection | 117–07 | |
| Chemical compound, drug | Calcium Phosphate transfection | This paper | https://www.nature.com/articles/nmeth0405-319 | Dr. Ryan Jensen laboratory. Material & Methods section |
| Chemical compound, drug | Lipofectamine3000 | Thermo Scientific | L3000001 | |
| Chemical compound, drug | TurboFect reagent | Thermo Scientific | R0533 | |
| Chemical compound, drug | G418 | American Bio | AB05058-00020 | |
| Chemical compound, drug | protease inhibitor cocktail | Sigma Aldrich | 04693159001 | |
| Chemical compound, drug | amylose resin | NEB | E8021 | |
| Chemical compound, drug | Hydroxyurea | Sigma Aldrich | 127-07-1 | |
| Chemical compound, drug | Benzonase | Millipore | 9025-65-4 | |
| Chemical compound, drug | MMC | Sigma Aldrich | M4287 | |
| Chemical compound, drug | Cisplatin | Sigma Aldrich | 15663-27-1 | |
| Chemical compound, drug | Olaparib | ApexBio | AZD2281, Ku-0059436 | |
| Chemical compound, drug | Talazoparib-BMN673 | ApexBio | A4153 | |
| Chemical compound, drug | DTT | Sigma Aldrich | 3483-12-3 | |
| Chemical compound, drug | IdU | Sigma Aldrich | I7125 | |
| Chemical compound, drug | CldU | Sigma Aldrich | C6891 | |
| Chemical compound, drug | FluorSave reagent | Calbiochem | 345789 | |
| Software, algorithm | ClustalIX | Larkin et al., 2007 | http://www.ch.embnet.org/software/ClustalW.html | |
| Software, algorithm | Bioedit | Bioedit (Hall, 1999) | https://itservices.cas.unt.edu/software/bioedit725 | |
| Software, algorithm | Snapgene 5.3.1. | Snapgene 5.3.1. | from Insightful Science; available at snapgene.com | |
| Software, algorithm | Prism Graph Pad version 9.0 | https://www.graphpad.com/updates/prism-900-release-notes | ||
| Software, algorithm | PyMOL Molecular Graphics System, Version 1.3 Schrödinger, LLC. | Schrodinger, L. (2010) The PyMOL Molecular Graphics System | ||
| Software, algorithm | Image J | Schneider et al., 2012 | https://imagej.nih.gov/ij/ | |
| Software, algorithm | ImageQuant TL 8.0 image analysis software | http://www.hhmi.umbc.edu/downloads/Imaging%20support%20GE/IQ%20TL%20collateral/IQTL_UserManual%208.pdf | ||
| Software, algorithm | Image Lab Software 6.1 (Bio-Rad) | https://www.bio-rad.com/en-us/product/image-lab-software?ID=KRE6P5E8Z&WT.mc_id=220411034041&WT.knsh_id=_kenshoo_clickid_&gclid=Cj0KCQjwr4eYBhDrARIsANPywCiJded2fEKfZWaf_-Fxki9ynO1VFgLb3kkN-gJ-A3u4baPwfvKj2ZQaApk-EALw_wcB | ||
| Software, algorithm | ChemiDoc MP imaging system XRS+ (Bio-Rad) | https://www.bio-rad.com/en-us/product/chemidoc-mp-imaging-system?ID=NINJ8ZE8Z&gclid=Cj0KCQjwr4eYBhDrARIsANPywCjmMNj5mMKTHZkXXj4iOrhnRLMo8JRt4nwLib3XTupvbHi2Q2CRJnkaAvp6EALw_wcB | ||
| Software, algorithm | Cell profiler software following counting and scoring pipeline | Carpenter et al., 2006 | ||
| Other | PDB structure BRC4 | Pellegrini et al., 2002 | 1NOW | Swiss model BRC2-7 |
| Other | DAPI stain | Thermo Fisher | D1306 | (1 µg/mL) |
| Other | NucRed Live 647 ReadyProbes Reagent | Thermo Fisher | R37106 | (1 µg/mL) |
