Journal: Cell Death & Disease

Article Title: Characterization of BRCA2 R3052Q variant in mice supports its functional impact as a low-risk variant

doi: 10.1038/s41419-023-06289-8

Figure Lengend Snippet: A Schematic representation of BRCA2 protein showing its functional domains and key binding partners. PALB2 binds to the N-terminal region of BRCA2, RAD51 binds to BRC repeats and the C-terminal region. There is N-terminal DNA binding (NTDB) domain spanning residues 265–341 and a C-terminal DNA binding (CTDB) domain spanning residues 2478–3185. The CTDB domain consists of an α-helical (αH) domain and three oligonucleotides/oligosaccharide bindings folds OB1-3. DSS1 is a small protein that binds to CTDB domain. The R3052Q/R2971Q variant lies at the junction of OB2 and OB3. B Representative images of mice of all the genotypes at weaning (21 days). C Body weight measurements of mice for 8 weeks post-weaning, mice of all genotypes show normal growth ( n = 10 animals, one-tailed t -test: two-sample assuming unequal variances, error bar—SE of mean). D H&E (upper panel), TUNEL (middle panel) and immunofluorescence images of spermatocyte spreads made from testes of 3-week old mice of indicated genotypes (lower panel). H&E staining reveals normal testis morphology in all the genotypes. TUNEL staining suggests lack of abnormal apoptosis in the testes of any genotype. Nuclease control (DNAseI treated) showed TUNEL positive staining. Spermatocyte spreads show chromosomes in late zygotene/pachytene stage labeled with SCP3 (red) and RAD51 (green) antibodies (upper panel scale bar = 200 μm, middle panel scale bar = 100 μm and lower panel scale bar = 5 μm). E Quantification of the number of RAD51 foci observed per spermatocyte colocalizing with SCP3 ( n > 30 spreads per genotype, error bar—SD of mean).

Article Snippet: Primary antibodies: BRCA2 (A303–434A; Bethyl Laboratories, 1:2000), Vinculin (sc-73614, Santa Cruz, 1:50,000), actin (I-19: sc-1616; Santa Cruz Biotechnology, 1:10,000) and Lamin B1 (12987-1-AP, Proteintech 1:50,000); diluted in blocking solution were used for overnight incubation of the membranes at 4 °C.

Techniques: Functional Assay, Binding Assay, Variant Assay, One-tailed Test, TUNEL Assay, Immunofluorescence, Staining, Control, Labeling

Journal: Cell Death & Disease

Article Title: Characterization of BRCA2 R3052Q variant in mice supports its functional impact as a low-risk variant

doi: 10.1038/s41419-023-06289-8

Figure Lengend Snippet: A Representative images of colony-forming assay of hematopoietic progenitor cells from fetal liver isolated from 16.5 dpc embryos of all genotypes with different concentration of olaparib. B Quantification of CFUs in untreated condition ( n = 3 biological replicates, * p < 0.05, ordinary one-way ANOVA, error bar—SE of mean). C Relative number of colonies formed from the fetal liver cells when exposed to increasing concentration of olaparib ( n = 3 biological replicates, one-tailed t -test: two-sample assuming unequal variances, error bar—SE of mean, *** p < 0.001). D Chromosomal aberrations in MEFs of indicated genotype in untreated (ctrl) and 100 nM MMC treated conditions (scale bar = 10 μm). Aberrations are marked with arrows. E Quantification of chromosomal aberrations in MEFs shown in ( D ). RQ/RQ and RQ/KO MEFs exhibit increased number of chromosomal aberrations after MMC treatment as compared to WT ( n > 24 spreads per genotype per treatment, two-way ANOVA, error bar—SD of mean, * p < 0.05, *** p < 0.001, **** p < 0.0001). F Western blot performed on whole cell lysate or total fraction (TF), cytoplasmic fraction (CF) and nuclear fraction (NF) of mES cells expressing WT and R3052Q BRCA2. Vinculin, Lamin B and Actin are used as CF, NF and TF loading controls, respectively. Graphical representation of amount of BRCA2 protein detected in the western blot normalized to Actin loading control ( n = 4 technical replicates (2 biological), * p < 0.05, Student’s t test, error bar—SE of mean).

Article Snippet: Primary antibodies: BRCA2 (A303–434A; Bethyl Laboratories, 1:2000), Vinculin (sc-73614, Santa Cruz, 1:50,000), actin (I-19: sc-1616; Santa Cruz Biotechnology, 1:10,000) and Lamin B1 (12987-1-AP, Proteintech 1:50,000); diluted in blocking solution were used for overnight incubation of the membranes at 4 °C.

Techniques: Isolation, Concentration Assay, One-tailed Test, Western Blot, Expressing, Control

Journal: Cell Death & Disease

Article Title: Characterization of BRCA2 R3052Q variant in mice supports its functional impact as a low-risk variant

doi: 10.1038/s41419-023-06289-8

Figure Lengend Snippet: Tumor incidence and spectrum of Brca2 RQ/+ , Brca2 RQ/RQ and Brca2 RQ/KO mice.

Article Snippet: Primary antibodies: BRCA2 (A303–434A; Bethyl Laboratories, 1:2000), Vinculin (sc-73614, Santa Cruz, 1:50,000), actin (I-19: sc-1616; Santa Cruz Biotechnology, 1:10,000) and Lamin B1 (12987-1-AP, Proteintech 1:50,000); diluted in blocking solution were used for overnight incubation of the membranes at 4 °C.

Techniques: Mouse Assay

Journal: Journal of International Society of Preventive & Community Dentistry

Article Title: BRCA1/2 Mutations in Salivary Pleomorphic Adenoma and Carcinoma-ex-Pleomorphic Adenoma

doi: 10.4103/jispcd.JISPCD_184_17

Figure Lengend Snippet: Higher magnification indicates BRCA2 nuclear positivity in myoepithelial cells and the cells in the chondroid material in pleomorphic adenoma samples

Article Snippet: Slides were incubated with primary mouse monoclonal anti-BRCA1 antibody (Abcam, ab16780) at 1:90 dilution and primary rabbit polyclonal anti-BRCA2 antibody (Abcam, ab 27976) at 1:80 for 1 h at room temperature, followed by incubation with secondary antibody for 30 min and immunostained with DAB (3, 3’-diaminobenzidine) for 5 min as a chromogen and hematoxylin as counterstain.

Techniques:

Journal: Journal of International Society of Preventive & Community Dentistry

Article Title: BRCA1/2 Mutations in Salivary Pleomorphic Adenoma and Carcinoma-ex-Pleomorphic Adenoma

doi: 10.4103/jispcd.JISPCD_184_17

Figure Lengend Snippet: A lower magnification indicates a strong BRCA2 positivity in polymorphous low-grade adenocarcinoma subtype. Notice the single-file cells and infiltrative invasive front

Article Snippet: Slides were incubated with primary mouse monoclonal anti-BRCA1 antibody (Abcam, ab16780) at 1:90 dilution and primary rabbit polyclonal anti-BRCA2 antibody (Abcam, ab 27976) at 1:80 for 1 h at room temperature, followed by incubation with secondary antibody for 30 min and immunostained with DAB (3, 3’-diaminobenzidine) for 5 min as a chromogen and hematoxylin as counterstain.

Techniques:

Journal: Nucleic Acids Research

Article Title: Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance

doi: 10.1093/nar/gkab308

Figure Lengend Snippet: Clustering of pathogenic BRCA2 missense mutations within the DBD. ( A ) Clinical significance of BRCA2 mutations by mutation type. The graph shows 9085 variants classified by molecular consequence (mutation type) and clinical significance, out of a total of 12 684 BRCA2 variants listed in the ClinVar database (version of 2020.07.27). ( B ) Clinical significance of 143 BRCA2 missense mutations reviewed by the expert panel are shown. Pathogenic mutations clustered in the DBD are marked with a blue circle. The schematic shows human BRCA2 protein structure. Amino acid residue numbers are marked. HD, helical domain: OB, oligonucleotide/oligosaccharide binding; TD, tower domain; NLS, nuclear localisation signal. ( C ) Location of each pathogenic mutation within the BRCA2 DBD.

Article Snippet: Primary antibodies used were α-BRCA2 (Ab-1, clone 2B, Merck Millipore OP95, 1:500; clone 5.23, Merck Millipore, 05-666, 1:2000), α-RAD51 (clone 14B4, GeneTex, GTX70230, 1:1000), α-GFP (clone JL-8, Clontech, 632381,1:1000), α-mCherry (α-dsRed, Clontech, 632496, 1:1000), α-Myc (clone 9E10, Santa Cruz, sc-40, 1:500), and α-MEK2 (BD Transduction, 610235, 1:1000).

Techniques: Mutagenesis, Residue, Binding Assay

Journal: Nucleic Acids Research

Article Title: Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance

doi: 10.1093/nar/gkab308

Figure Lengend Snippet: Pathogenic BRCA2 mutations in the DBD affect DSS1 binding and nuclear localization. ( A ) List of pathogenic (red) or benign (blue) mutations in the DBD analysed in this study with a schematic of the expression construct, GFP-BRCA2 CT . ( B and C ) Interaction between the DBD mutants and DSS1. Wild-type or mutant forms of GFP-BRCA2 CT were transiently expressed with Myc-DSS1 in 293T cells. Interaction was detected by anti GFP-IP and western blotting with anti-Myc antibody. The graph ( C ) shows DSS1-binding activity of the mutants normalised to the WT value. Binding activity was assessed by quantifying the intensity of the Myc-DSS1 bands pulled-down by IP. Data are presented as mean ± s.e. from three repeats. Statistical significance was assessed by 1-way ANOVA test, followed by Dunnett's multiple comparison test (compared to WT). (D and E) Nucleocytoplasmic distribution of the DBD mutants. HeLa cells transiently expressing GFP-BRCA2 CT fragments were fixed with PFA before imaging. The graph ( E ) shows the GFP intensity ratio (nuclear/cytoplasmic, mean ± s.e.). More than 100 cells per sample were analysed. Statistical significance was assessed by the Kruskal-Wallis test, followed by Dunnett's multiple comparison test (compared to WT). A representative result from two independent experiments is shown. ( D ) shows representative microscopic images. Scale bar, 10μm.

Article Snippet: Primary antibodies used were α-BRCA2 (Ab-1, clone 2B, Merck Millipore OP95, 1:500; clone 5.23, Merck Millipore, 05-666, 1:2000), α-RAD51 (clone 14B4, GeneTex, GTX70230, 1:1000), α-GFP (clone JL-8, Clontech, 632381,1:1000), α-mCherry (α-dsRed, Clontech, 632496, 1:1000), α-Myc (clone 9E10, Santa Cruz, sc-40, 1:500), and α-MEK2 (BD Transduction, 610235, 1:1000).

Techniques: Binding Assay, Expressing, Construct, Mutagenesis, Western Blot, Activity Assay, Comparison, Imaging

Journal: Nucleic Acids Research

Article Title: Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance

doi: 10.1093/nar/gkab308

Figure Lengend Snippet: The BRCA2-DSS1 interaction antagonizes intracellular BRCA2 oligomerization. ( A ) Self-oligomerization of BRCA2 CT is suppressed by Myc-DSS1. Wildtype or D2723H mutant forms of BRCA2 CT , tagged with either GFP or mCherry, were transiently expressed in 293T cells with or without Myc-DSS1. Interaction was detected by anti GFP-IP and western blotting for mCherry-BRCA2 CT and endogenous BRCA2. ( B ) Self-oligomerization of BRCA2 CT fragments in the BRCA2 Knock-out (KO) HeLa cells. BRCA2 CT fragments tagged with either GFP or mCherry were co-expressed in BRCA2 KO HeLa cells. Their interaction was detected by anti GFP-IP and western blotting for mCherry-BRCA2 CT . GFP vector alone serves as a negative control. ( C ) GST-pull down assay. Purified GST-BRCA2 CT fragments bound to the Glutathione Sepharose 4B beads were incubated with purified GFP-BRCA2 CT fragments. The beads were washed, and bound proteins were eluted, before separation by SDS-PAGE. Interactions were detected by western blotting for GFP-BRCA2 CT . GST alone serves as a negative control. ( D ) Native gel electrophoresis of GFP-BRCA2 CT . Extracts from HeLa cells transiently expressing GFP-BRCA2 CT forms with or without Myc-DSS1 were analysed by native gel electrophoresis. The bands corresponding to GFP-BRCA2 CT oligomers, GFP-BRCA2 CT /Myc-DSS1 complex and GFP-BRCA2 CT are marked with arrows.

Article Snippet: Primary antibodies used were α-BRCA2 (Ab-1, clone 2B, Merck Millipore OP95, 1:500; clone 5.23, Merck Millipore, 05-666, 1:2000), α-RAD51 (clone 14B4, GeneTex, GTX70230, 1:1000), α-GFP (clone JL-8, Clontech, 632381,1:1000), α-mCherry (α-dsRed, Clontech, 632496, 1:1000), α-Myc (clone 9E10, Santa Cruz, sc-40, 1:500), and α-MEK2 (BD Transduction, 610235, 1:1000).

Techniques: Mutagenesis, Western Blot, Knock-Out, Plasmid Preparation, Negative Control, Pull Down Assay, Purification, Incubation, SDS Page, Nucleic Acid Electrophoresis, Expressing

Journal: Nucleic Acids Research

Article Title: Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance

doi: 10.1093/nar/gkab308

Figure Lengend Snippet: Pathogenic DBD mutants introduced into the endogenous BRCA2 gene cause cytosolic mis-localization and disable DNA repair by HDR. ( A ) Subcellular fractionation of BRCA2 mutant cell lines, HeLa-W2626C and HeLa-D2723H. Two independent clones for each mutation were used. Nuclear and cytoplasmic fractions of BRCA2 and RAD51 were analysed by western blotting. MEK2 is a cytoplasmic marker. ( B ) mClover Lamin A HDR assay. Cells transfected with Lamin A-targeting sgRNA and mClover Lamin A donor constructs as shown in the schematic were analysed for mClover Lamin A-positive cells. Mean of HDR positive cells (%) ± s.e. from two repeats is shown. More than 100 cells per sample were analysed in each repeat. Statistical significance was tested by one-way ANOVA test, followed by Bonferroni's multiple comparison test.

Article Snippet: Primary antibodies used were α-BRCA2 (Ab-1, clone 2B, Merck Millipore OP95, 1:500; clone 5.23, Merck Millipore, 05-666, 1:2000), α-RAD51 (clone 14B4, GeneTex, GTX70230, 1:1000), α-GFP (clone JL-8, Clontech, 632381,1:1000), α-mCherry (α-dsRed, Clontech, 632496, 1:1000), α-Myc (clone 9E10, Santa Cruz, sc-40, 1:500), and α-MEK2 (BD Transduction, 610235, 1:1000).

Techniques: Fractionation, Mutagenesis, Clone Assay, Western Blot, Marker, Transfection, Construct, Comparison

Journal: Nucleic Acids Research

Article Title: Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance

doi: 10.1093/nar/gkab308

Figure Lengend Snippet: Pathogenic DBD mutants exhibit defective nuclear import. ( A ) Nuclear import assay using BRCA2 CT tagged with photoactivable GFP (paGFP). HeLa cells expressing wildtype or mutant forms of paGFP-BRCA2 CT were monitored by live imaging. As shown in the schematic, photoactivation of a cytosolic area (red square) was followed by measurement of fluorescence intensity in the designated area in the cytosol (red square) and the nucleus (blue square). Plots show the relative fluorescence intensity (mean ± s.e.), normalized to the maximum intensity in each cell. 8–10 cells per sample were analysed. Black arrows mark the timing of photoactivation. ( B ) HeLa cells expressing paGFP-BRCA2 CT W2626C with or without Myc-DSS1 were monitored by live imaging after cytosolic photoactivation and analysed as shown in (A). ( C ) Efficiency of DSS1 depletion by siRNA transfection. Relative level of DSS1 mRNA measured by RT-qPCR assay, a result from technical triplicate is shown. Statistical significance was assessed by paired t -test. ( D and E ) Nucleocytoplasmic distribution of the GFP-BRCA2 CT fragment after DSS1 depletion. HeLa cells were transfected with control or DSS1 siRNA 24 h before transfecting with the GFP-BRCA2 CT construct. Nucleocytoplasmic distribution was analysed as described in Figure . The graph (E) shows the GFP intensity ratio (nuclear/cytoplasmic, mean ± s.e.). More than 100 cells per sample were analysed. Statistical significance was assessed by unpaired t-test. A representative result from three independent experiments is shown. Representative microscopic images are shown in (D). Scale bar, 10 μm. ( F ) Subcellular fractionation of HeLa cells after DSS1 depletion. HeLa cells were transfected with control or DSS1 siRNA 48 h before fractionation. Nuclear and cytoplasmic fractions of BRCA2 and RAD51 were analysed by western blotting. The nuclear/cytoplasmic ratio of BRCA2 expression is shown. MEK2 is a cytoplasmic marker. Total cell extracts are also shown. A representative result from two independent experiments is shown.

Article Snippet: Primary antibodies used were α-BRCA2 (Ab-1, clone 2B, Merck Millipore OP95, 1:500; clone 5.23, Merck Millipore, 05-666, 1:2000), α-RAD51 (clone 14B4, GeneTex, GTX70230, 1:1000), α-GFP (clone JL-8, Clontech, 632381,1:1000), α-mCherry (α-dsRed, Clontech, 632496, 1:1000), α-Myc (clone 9E10, Santa Cruz, sc-40, 1:500), and α-MEK2 (BD Transduction, 610235, 1:1000).

Techniques: Expressing, Mutagenesis, Imaging, Fluorescence, Transfection, Quantitative RT-PCR, Control, Construct, Fractionation, Western Blot, Marker

Journal: Nucleic Acids Research

Article Title: Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance

doi: 10.1093/nar/gkab308

Figure Lengend Snippet: DSS1 expression restores the nuclear localization and HDR function of hypomorphic BRCA2 pathogenic mutants. ( A and B ) Nucleocytoplasmic distribution of the DBD mutants with DSS1 overexpression. HeLa cells expressing GFP-BRCA2 CT forms without (vector only) or with Myc-DSS1 were fixed with PFA and analysed by microscopy. Representative images are shown. Scale bar, 10μm. The graph (B) shows GFP intensity ratio (nuclear/cytoplasmic, mean ± s.e.) measured from the images. Myc-DSS1 expression increases the nuclear/cytoplasmic ratio of GFP-BRCA2 CT fragments to varying degrees: WT by 1.7 fold; W2626C and E2663V mutants by 3.5- to 5-fold; I2627F, R2659T, and G2748D mutants by 1.8- to 2.4-fold; T2722R, D2723H, and D2723G mutants, little change. More than 100 cells per sample were analysed. Statistical significance was assessed by one-way ANOVA test, followed by Bonferroni's multiple comparison test. A representative result from three independent experiments is shown. ( C ) mClover Lamin A HDR assay. HeLa cells harbouring BRCA2 mutations (Figure ) were transfected with vector or Myc-DSS1 plasmids in addition to mClover Lamin A HDR assay plasmids. Mean of HDR positive cells (%) ± s.e. from two repeats is shown. More than 100 cells per sample were analysed in each repeat. Statistical significance was assessed by one-way ANOVA test, followed by Bonferroni's multiple comparison test.

Article Snippet: Primary antibodies used were α-BRCA2 (Ab-1, clone 2B, Merck Millipore OP95, 1:500; clone 5.23, Merck Millipore, 05-666, 1:2000), α-RAD51 (clone 14B4, GeneTex, GTX70230, 1:1000), α-GFP (clone JL-8, Clontech, 632381,1:1000), α-mCherry (α-dsRed, Clontech, 632496, 1:1000), α-Myc (clone 9E10, Santa Cruz, sc-40, 1:500), and α-MEK2 (BD Transduction, 610235, 1:1000).

Techniques: Expressing, Over Expression, Plasmid Preparation, Microscopy, Comparison, Transfection

Journal: Nucleic Acids Research

Article Title: Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance

doi: 10.1093/nar/gkab308

Figure Lengend Snippet: Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance. Our results suggest a hypothetical model wherein the engagement of DSS1 by the DBD of BRCA2 antagonizes BRCA2 self-oligomerization and promotes protein folding. Assembly of a correctly folded BRCA2/DSS1 complex enables nuclear transport. Cancer-causing mutations in the BRCA2 DBD impair DSS1 binding, perturbing this protein assembly mechanism, and provoking the formation of intracellular BRCA2 oligomers that are excluded from the cell nucleus. The failure to assemble correctly folded mutant BRCA2/DSS1 complexes, and cytosolic mis-localization of the mutant BRCA2, disables HDR in cells harbouring the DBD mutations. DSS1 can antagonize the self-oligomerization of wild-type BRCA2, and of cancer-causing DBD mutants such as W2626C that retain some degree of DSS1 binding.

Article Snippet: Primary antibodies used were α-BRCA2 (Ab-1, clone 2B, Merck Millipore OP95, 1:500; clone 5.23, Merck Millipore, 05-666, 1:2000), α-RAD51 (clone 14B4, GeneTex, GTX70230, 1:1000), α-GFP (clone JL-8, Clontech, 632381,1:1000), α-mCherry (α-dsRed, Clontech, 632496, 1:1000), α-Myc (clone 9E10, Santa Cruz, sc-40, 1:500), and α-MEK2 (BD Transduction, 610235, 1:1000).

Techniques: Binding Assay, Mutagenesis

Journal: Nature Communications

Article Title: Elongator and codon bias regulate protein levels in mammalian peripheral neurons

doi: 10.1038/s41467-018-03221-z

Figure Lengend Snippet: Depleted levels of BRCA2 are correlated with elevated levels of DNA damage. a RT-qPCR confirms normal levels of Brca2 transcript ( P = 0.06). b – d Quantitative immunohistochemistry confirms decreased levels of BRCA2 protein ( P < 0.001). e – g Longer comet tails in the CKO show fragmented DNA ( P = 0.009); error bars denote SD in a and SEM in d , g ; scale bar: b , c 40 μm; e , f 12 μm

Article Snippet: Primary antibodies included the following: BRCA2 (Biorbyt, orb10203, 1:25), VCAN (Abcam, ab177480, 1:100), S-100 (Agilent Technologies, Z0311, 1:400), Tuj1 (Biolegend, 801202, 1:1,000), and Histone H2A (Biorbyt, orb127582, 1:200).

Techniques: Quantitative RT-PCR, Immunohistochemistry

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Low-Dose Ionizing Radiation Affects Mesenchymal Stem Cells via Extracellular Oxidized Cell-Free DNA: A Possible Mediator of Bystander Effect and Adaptive Response

doi: 10.1155/2017/9515809

Figure Lengend Snippet: DNA damage induced by low-dose radiation and cfDNAox and cfDNAoxR fragments activates reparation of nuclear DNA. (a) (1) Flow cytometry detection of BRCA2 in irradiated (10 cGy) and control cells stained with anti-BRCA2 antibodies and secondary FITC-conjugated antibodies. Gate R encircles the fraction of MSCs with elevated values of FL1- BRCA2; (2) distribution of cells with varying BRCA2 contents. (b) The signal intensity of FL1-R (1) and average signal intensity of FL1-BRCA2 (2) in irradiated cells (10 cGy, 15 min and 2 h after exposure) and cells exposed to cfDNAox and cfDNAoxR (50 ng/ml, 15 min and 2 h) (flow cytometry). (c) Linear correlation between the levels of γ H2AX and BRCA2 ( k = 0.96; p < 0.0001). (d, e) Dependence of the changes in the levels of mRNA BRCA1 (d) and BRCA2 (e) in irradiated cells and cells exposed to cfDNAox and cfDNAoxR (RT-PCR); mRNA level—average expression of genes in treated cells compared to control (for three biological replicates). Reference gene, TBP . ∗ p < 0.001, nonparametric U test (qRT-PCR).

Article Snippet: The following antibodies were used: γ H2AX-Dylight488 (pSer139) (NB100-78356G, Novus Biologicals); NOX4 (Sc-30141, Santa Cruz Biotechnology); 8OHDG (Sc-66036, Santa Cruz Biotechnology); BRCA2 (NBP1-88361, Novus Biologicals); PCNA (ab2426, Abcam); Ki-67FITC (sc-23900 FITC, Santa Cruz Biotechnology); and BCL2 (Sc-783, Santa Cruz Biotechnology).

Techniques: Flow Cytometry, Irradiation, Control, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Sulphostin-inspired N -phosphonopiperidones as selective covalent DPP8 and DPP9 inhibitors

doi: 10.1038/s41467-025-58493-z

Figure Lengend Snippet: a X-ray structure of 16 in complex with DPP9 refined to a final resolution of 2.49 Å, shown from two different angles. The ligand is covalently bound to the catalytically active serine S730 residue and is contoured at 1 σ with the refined 2F o -F c electron density map. Hydrogen bond interactions are depicted as dashed lines. b ABPP without (FC plotted on x-axis) or after pre-treatment with 50 µM 8, 11 or 16 (competition experiment, FC plotted on y-axis) with 10 µM 9 after click chemistry ( n = 4 biologically independent samples). Dashed lines indicate the FC ≥ 2 ( 9 /DMSO) or FC ≤ -2 (Competitor/ 9 ) threshold. Black symbols indicate identified protein groups. To identify statistically significant hits from the analysis (marked as a triangle or square), p ≤ 0.01 (two-sided Student’s t -test, permutation-based FDR with 250 randomizations and FDR = 0.05) was applied. c Addition of N -phosphono - ( S )−3-aminopiperidine-2-ones block the interaction between endogenous DPP9 and BRCA2. Quantification of PLAs showing MMC-induced DPP9-BRCA2 PLA events in HeLa wild-type cells, in the presence of 10 µM of the respective inhibitory compounds. Cells were treated with 300 nM MMC for 24 h and 10 µM of the indicated inhibitors for 1 h prior to fixation. Each dot represents the number of PLA events in a single cell. Data were analyzed by unpaired two-sided t -test comparisons (**** p < 0.0001). To visualize the three biological replicates, each biological replicate is labeled differently: circle, triangle or square. n values indicate total number of cells in each analysis group from 3 independent biological replicates in total: n = 240 for BRCA2 Ab-Ctrl., n = 237 for DPP9 Ab-Ctrl., n = 222 for + Ctrl., n = 202 for 1G244, n = 231 for 8, n = 205 for 16 . d Inhibition of DPP8/9 increases cellular sensitivity to genotoxic stress. HeLa wild-type (WT) cells were treated with 1 µM MMC and 10 µM of the respective inhibitors. Cell viability was measured after 72 h, and normalized to WT mock treated cells. DPP9 KO cells and WT cells treated with 1G244 (10 µM) were analyzed as positive controls, whereas WT cells treated with Sitagliptin were used as a negative control. Dashed lines indicate the viability of the WT + MMC cells. The graph shows the mean and error bars indicate the SEM of all individual measurements ( n = 18 (HeLa wild-type controls), 17 (HeLa DPP9 KO control) or 9 (inhibitor-treated samples) independent biological replicates). Data were analyzed by a Tukey two-way ANOVA, using a mixed effect analysis (n.s. = not significant, * p = 0.0157, **** p < 0.0001). Source data are provided as a Source Data file.

Article Snippet: Cells were incubated with primary antibodies against BRCA2 (R&D Systems, USA, Cat# MAB2476, RRID:AB_2259370, 1:100) and DPP9 (Ruth Geiss-Friedlander—University of Freiburg Cat# RGF_1, RRID:AB_2889071, 1:100) for 90 min at 37 °C and actin filaments were simultaneously counterstained with CytoPainter Phalloidin‐iFluor 488 Reagent (Abcam, United Kingdom).

Techniques: Residue, Blocking Assay, Labeling, Inhibition, Negative Control, Control

Journal: bioRxiv

Article Title: Synergistic effects of PARP inhibitors by Schlafen 11 and BRCA2-deficiency through accumulation of single-strand DNA gaps behind a fork

doi: 10.1101/2023.06.28.546820

Figure Lengend Snippet: SLFN11 synergistically enhanced sensitivity to PARP inhibitors in BRCA2-deficient cells. A , Immunoblot analysis of whole cell lysates prepared from TOV-112D cells: SLFN11-proficient (parent), SLFN11-KO, control siRNA (siCON) or BRCA2 siRNA (siBRCA2). Blots were probed with anti-SLFN11 and anti-BRCA2 antibodies. Tubulin was used as a loading control. B , Viability of TOV-112D cells under each condition atier 48 h of continuous CPT treatment. Cellular ATP activity was used to measure cell viability. The viability of untreated cells was set as 100%. Data are means ± standard deviations (n = 3, technical replicates). C , Viability of TOV-112D cells under each condition atier 48 h of continuous treatment with PARP inhibitors. Cellular ATP activity was used to measure cell viability. The survival of untreated cells was set as 100%. Data are means ± standard deviations (n = 3, technical replicates). D , Apoptosis analysis of TOV-112D cells by flow cytometry treated continuously with 2.5 μM olaparib for 0, 24, or 48 h. NS: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance with Tukey’s post-hoc multiple comparisons test).

Article Snippet: Antibodies against SLFN11 (cat. no. sc-515071) and CHK1 (cat. no. sc-8408) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against BRCA2 (cat. no. 10741), RPA2 (cat. no. 35869), histone H3 (cat. no. 4499), PAR (cat. no. 83732), PARP1 (cat. no. 9532), phospho-CHK1 (S345) (cat. no. 2348) were from Cell Signaling Technology (Danvers, MA, USA); and antibodies against α-tubulin (cat. no. 017-25031) were from Wako.

Techniques: Western Blot, Control, Activity Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Synergistic effects of PARP inhibitors by Schlafen 11 and BRCA2-deficiency through accumulation of single-strand DNA gaps behind a fork

doi: 10.1101/2023.06.28.546820

Figure Lengend Snippet: SLFN11 and BRCA2-deficiency increased chromatin-bound RPA2 under PARPis. A , Immunoblot analysis of chromatin-bound fractions prepared from TOV-112D cells. Cells were treated with 10 μM olaparib for 0, 6, or 12 h. Blots were probed with anti-RPA2 antibodies. Histone H3 was used as a loading control. B , Quantification of data from A . Data were normalized to the untreated cells (every 0 h). Data are means ± standard deviations (n = 3, biological replicates). C , Representative confocal microscopy images; chromatin-bound RPA2 (red) and Hoechst (blue) in TOV-112D cells. Cells were treated with or without 10 μM olaparib for 12 h. D , Quantification of data from C . Scatier plots show the mean signal intensity of RPA2. Data are means ± standard deviations (n = 116–199, one-time experiment). NS: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance with Tukey’s post-hoc multiple comparisons test).

Article Snippet: Antibodies against SLFN11 (cat. no. sc-515071) and CHK1 (cat. no. sc-8408) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against BRCA2 (cat. no. 10741), RPA2 (cat. no. 35869), histone H3 (cat. no. 4499), PAR (cat. no. 83732), PARP1 (cat. no. 9532), phospho-CHK1 (S345) (cat. no. 2348) were from Cell Signaling Technology (Danvers, MA, USA); and antibodies against α-tubulin (cat. no. 017-25031) were from Wako.

Techniques: Western Blot, Control, Confocal Microscopy

Journal: bioRxiv

Article Title: Synergistic effects of PARP inhibitors by Schlafen 11 and BRCA2-deficiency through accumulation of single-strand DNA gaps behind a fork

doi: 10.1101/2023.06.28.546820

Figure Lengend Snippet: SLFN11 and BRCA2-deficiency increased ssDNA gaps in the presence of PARPis. A , Scheme of alkaline BrdU comet assay. TOV-112D cells under each condition were treated with or without drug for 6 h, labeled with BrdU for 30 min during that time, and then incubated in BrdU-free medium for the last 90 min. Atier electroporation under alkaline conditions, tail moments of BrdU-labeled cells were measured. B , Representative alkaline BrdU comet assay images in TOV-112D cells treated with or without 10 μM olaparib. C , E , F , scatier plots show BrdU tail moments in TOV-112D cells under each drug treatment ( C : 10 μM olaparib, E : 100 nM olaparib, F : 100 nM CPT). Data are means ± standard deviations ( C : n = 54–59, E : n = 54–77, F : n = 91–139, one-time experiment). D , Immunoblot analysis of PAR levels in whole cell lysates from parent TOV-112D cells treated as indicated for 30 min. Blots were probed with anti-PAR antibodies. The asterisk indicates a nonspecific band. Tubulin was used as a loading control. NS: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance with Tukey’s post-hoc multiple comparisons test).

Article Snippet: Antibodies against SLFN11 (cat. no. sc-515071) and CHK1 (cat. no. sc-8408) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against BRCA2 (cat. no. 10741), RPA2 (cat. no. 35869), histone H3 (cat. no. 4499), PAR (cat. no. 83732), PARP1 (cat. no. 9532), phospho-CHK1 (S345) (cat. no. 2348) were from Cell Signaling Technology (Danvers, MA, USA); and antibodies against α-tubulin (cat. no. 017-25031) were from Wako.

Techniques: Single Cell Gel Electrophoresis, Labeling, Incubation, Electroporation, Western Blot, Control

Journal: bioRxiv

Article Title: Synergistic effects of PARP inhibitors by Schlafen 11 and BRCA2-deficiency through accumulation of single-strand DNA gaps behind a fork

doi: 10.1101/2023.06.28.546820

Figure Lengend Snippet: BRCA2-deficiency enhanced the recruitment of SLFN11 on chromatin under PARPis. A , Immunoblot analysis of chromatin-bound fractions prepared from TOV-112D cells. Cells were treated with 10 μM olaparib for 0, 4, 8, or 12 h. Blots were probed with anti-RPA2, anti-SLFN11, and anti-PAPR1 antibodies. Histone H3 was used as a loading control. B , Quantification of data from A . Data were normalized to untreated cells (every 0 h). Data are means ± standard deviations (n = 5–7, biological replicates). C , Immunoblot analysis of whole cell lysates prepared from TOV-112D cells. Blots were probed with anti-SLFN11 and anti-BRCA2 antibodies. Tubulin was used as a loading control. D , Representative confocal microscopy images; Hoechst (blue), chromatin-bound RPA2 (red), and SLFN11 (green) in TOV-112D cells. Cells were treated with or without 10 μM olaparib for 12 h. Representative tracings of the distribution of signals along the white dashed arrow ( a and b ) are shown in the merged panel. E , Quantification of data from D . Scatier plots show mean signal intensities of RPA2 and SLFN11. Data are means ± standard deviations (n = 104–195, one-time experiment). NS: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance with Tukey’s post-hoc multiple comparisons test).

Article Snippet: Antibodies against SLFN11 (cat. no. sc-515071) and CHK1 (cat. no. sc-8408) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against BRCA2 (cat. no. 10741), RPA2 (cat. no. 35869), histone H3 (cat. no. 4499), PAR (cat. no. 83732), PARP1 (cat. no. 9532), phospho-CHK1 (S345) (cat. no. 2348) were from Cell Signaling Technology (Danvers, MA, USA); and antibodies against α-tubulin (cat. no. 017-25031) were from Wako.

Techniques: Western Blot, Control, Confocal Microscopy

Journal: bioRxiv

Article Title: Synergistic effects of PARP inhibitors by Schlafen 11 and BRCA2-deficiency through accumulation of single-strand DNA gaps behind a fork

doi: 10.1101/2023.06.28.546820

Figure Lengend Snippet: Resection by MRE11 was required for SLFN11 to be recruited on chromatin. A , Immunoblot analysis of chromatin-bound fractions prepared from TOV-112D (SLFN11-KO) cells. Cells were treated with 10 μM olaparib and with or without 50 μM mirin for 0, 6, or 12 h. Blots were probed with anti-RPA2 antibodies. Histone H3 was used as a loading control. B , Quantification of data from A . Data were normalized to untreated cells (every 0 h). Data are means ± standard deviations (n = 3–4, biological replicates). C , Immunoblot analysis of chromatin-bound fractions prepared from TOV-112D (parent) cells. Cells were treated with 10 μM olaparib and with or without 50 μM mirin for 0, 6, or 12 h. Blots were probed with anti-RPA2 and anti-SLFN11 antibodies. Histone H3 was used as a loading control. D , Quantification of data from C . Data were normalized to untreated cells (every 0 h). Data are means ± standard deviations (n = 3–4, biological replicates). E , Schematic illustration of the working model. (1) CPT treatment, (2) PARPi treatment. (1) CPT (or a typical DNA damaging agent) exerts replication stress, where the helicase and polymerase become uncoupled, causing the formation of RPA-coated ssDNA gaps in between. SLFN11 binds the gaps on forks and blocks replication. (2-1) In SLFN11-deficient/BRCA2-proficient cells, PARPis generate PARP-trapping, and the DNA strand progressing toward the pre-existing Okazaki fragment is resected by MRE11 due to impaired OFP. The ssDNA gap length or the number of resection sites is limited compared with that under other conditions, and the sensitivity to PARPis is also limited. (2-2) In SLFN11-deficient/BRCA2-deficient cells, ssDNA gaps increase more than in SLFN11-deficient/BRCA2-proficient cells. Moreover, OFP impairment induced by BRCA2-deficiency should contribute to the accumulation of ssDNA gaps. (2-3) In SLFN11-proficient/BRCA2-proficient cells, SLFN11 is recruited to ssDNA gaps atier resection by MRE11 and contributes to the accumulation of ssDNA gaps. (2-4) BRCA2-deficiency causes more ssDNA gaps, as explained in (2-2), where SLFN11 recruitment is enhanced. Consequently, PARPis show the most accumulation of ssDNA gaps in BRCA2-deficient/SLFN11-proficient cells and exhibit the highest anticancer effects. See Discussion for details. NS: not significant, * P < 0.05, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance with Tukey’s post-hoc multiple comparisons test).

Article Snippet: Antibodies against SLFN11 (cat. no. sc-515071) and CHK1 (cat. no. sc-8408) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against BRCA2 (cat. no. 10741), RPA2 (cat. no. 35869), histone H3 (cat. no. 4499), PAR (cat. no. 83732), PARP1 (cat. no. 9532), phospho-CHK1 (S345) (cat. no. 2348) were from Cell Signaling Technology (Danvers, MA, USA); and antibodies against α-tubulin (cat. no. 017-25031) were from Wako.

Techniques: Western Blot, Control