antibodies against brca2  (R&D Systems)

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: Nature Communications

Article Title: CGMega: explainable graph neural network framework with attention mechanisms for cancer gene module dissection

doi: 10.1038/s41467-024-50426-6

Figure Lengend Snippet: a Scatter plot shows the gene participation in cancer gene modules. In gene modules of 358 well-known breast cancer genes, 22 known cancer genes (blue dots) and another 12 genes (red dots) which were not known as breast cancer genes were highly involved (participated in over 20 cancer gene modules). Gray dots donated known cancer genes which were not highly involved in cancer gene modules. b In total, 347 positive cancer genes of breast cancer were generally divided into five clusters (by K-means clustering) based on feature importance scores. c An example for illustrating gene representation features (RFs). For a given gene, if a feature is assigned with an importance score (calculated by GNNExplainer) 10 times higher than the minimum score, it will be referred to as the RF of this gene. d Illustrations of BRCA1 and BRCA2 gene modules. e Western Blot analysis after 24 h, 48 h, and 72 h treatment. Each experiment was repeated three times independently. f , g Half maximal inhibitory concentration (IC 50 ) value of olaparib treatment ( f ) and olaparib/RKI-1447 combination treatment ( g ) after 24 hr. h The inhibition rate of olaparib combined with RKI-1447 is significantly higher than that of olaparib alone after 24 h treatment. Paired t test of two-sided was used to analyze the mean inhibition rates from two groups and the P value = 0.0023. f – h Data are presented as mean values +/− SEM and n = 3 biologically independent experiments were carried out for each to derive statistics. Source data are provided as a Source Data file.

Article Snippet: The membranes containing proteins were incubated at 4 °C for 16 h with primary antibodies against BRCA2 (1:500, EPR23442-43, Abcam, Cambridge, United Kingdom, Cat No 29450-1-AP, clone name: FACD, FANCD1), ROCK2 (1:1000, sc-100425, SANTA CRUZ Biotechnology, the United States, Cat No 21645-1-AP, clone name: KIAA0619, p164 ROCK2, Rho kinase 2), GAPDH (1:30,000, 80570-1-RR, Proteintech, the United States) and then with secondary HRP-conjugated anti-mouse (1:3000, A0216, Beyotime, China), or anti-rabbit antibodies (1:3000, A0208, Beyotime, China) for 1 h at room temperature.

Techniques: Western Blot, Concentration Assay, Inhibition

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

Journal: bioRxiv

Article Title: RNA transcripts suppress G-quadruplex structures through G-loop formation

doi: 10.1101/2023.03.09.531892

Figure Lengend Snippet: a, Mock-, RAD51-, and BRCA2-depleted NPEs were analysed by Western blot with RAD51 and BRCA2 antibodies. b, pG4 BOT was incubated in the NPEs described in a , and products were analysed by DRIP-qPCR using primers for the G4 locus. Where indicated, NPE was pre-treated with RNaseA. Relative values compared to input signals were plotted. c , Scheme of the chromatin immunoprecipitation (ChIP) assay. G4-containing plasmids are incubated in NPE, DNA and proteins (ovals) are crosslinked, sonicated, and immunoprecipitated with the antibody of interest (magenta). The co-precipitated DNA is amplified by quantitative PCR (qPCR) with primers (magenta arrows) specific to the G4 locus. d, pdsG4 BOT , pG4 BOT , and pG4 DBL were incubated in NPE, and products were analysed by ChIP-qPCR with RAD51, BRCA2, and hnRNPA1 antibodies and a primer pair for the G4 locus. The relative values compared to the highest signal among the conditions were plotted. e, Mock- and hnRNPA1-depleted NPEs supplemented with buffer or the hnRNPA1 complex purified from extract, were analysed by Western blot with the hnRNPA1 antibody. f, pG4 BOT was incubated in the NPEs described in e , and products were analysed by DRIP-qPCR as in b . Where indicated, NPE was pre-treated with RNaseA.

Article Snippet: Antibodies against xl BRCA2 , xl DHX36 , xl FANCD2 , xl FANCJ , xl MUS81 , hs RAD51 , xl SLX1 , xl SLX4 , xl XPA , xl XPF and hs RNA Polymerase II subunit A (Bethyl) were previously described.

Techniques: Western Blot, Incubation, Chromatin Immunoprecipitation, Sonication, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, ChIP-qPCR, Purification

Journal: EMBO Molecular Medicine

Article Title: Thioparib inhibits homologous recombination repair, activates the type I IFN response, and overcomes olaparib resistance

doi: 10.15252/emmm.202216235

Figure Lengend Snippet: A Structures of thioparib and its enantiomer Cpd‐391. B The concentration‐effect relationships of PARP1 inhibition by thioparib, talazoparib, and olaparib, as assayed by histone‐based ELISA. Data are obtained from three biological replicates and depicted as mean ± SEM. C Concentration‐dependent increase in PARP1‐DNA binding by three PARP inhibitors using DSB FA assays. FA was measured 60 min after adding NAD + , and the ΔFA values represent the changes in PARP1‐DNA binding. Data from four biological replicates are presented as mean ± SEM. D Correlation between cytotoxicity and EC 50 , as measured by the DSB FA model. The cytotoxicity data are shown in Appendix Table . The Pearson test was performed to calculate the correlation. E IC 50 values of the 13 isoforms of PARPs, as detected by biotinylated NAD + ‐based luminescence assays. Numbers with superscripts are data from the reference (Antolin et al , ). F, G Effects of thioparib on BRCA1‐deficient MDA‐MB‐436 (F) and BRCA2‐deficient Capan‐1 (G) xenografts. Mice‐bearing MDA‐MB‐436 tumors were dosed orally with 10 mg/kg thioparib, 100 mg/kg olaparib, or vehicle (Veh) once daily for 3 weeks; and mice‐bearing Capan‐1 tumors were dosed orally with 5 or 25 mg/kg thioparib, 0.3 mg/kg talazoparib, or vehicle (Veh) once daily for 3 weeks ( n = 6). Data are shown as mean ± SEM. Statistical analysis was performed by two‐way ANOVA. *** P < 0.0001. Representative images of xenograft tumors are shown, and mice tails represent complete regression of tumors. Data information: ThP, thioparib; OP, olaparib; TP, talazoparib; RP, rucaparib; VP, veliparib; 391, Cpd‐391.

Article Snippet: Antibody against BRCA1 (OP92) and BRCA2 (OP95) were from Millipore.

Techniques: Concentration Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: EMBO Molecular Medicine

Article Title: Thioparib inhibits homologous recombination repair, activates the type I IFN response, and overcomes olaparib resistance

doi: 10.15252/emmm.202216235

Figure Lengend Snippet: Thioparib and olaparib sensitivity of WT and 53BP1#KO (53BP1 −/− ) MDA‐MB‐436 cells. Cells were treated with thioparib or olaparib for 7 d and measured by CCK8 assay. Data from three independent experiments are presented as mean ± SEM. Efficacy of thioparib in the PARPi‐resistant CDX model MDA‐MB‐436 53BP1#KO. Mice‐bearing tumors ( n = 6) were dosed with 10 mg/kg thioparib, 100 mg/kg olaparib, 10 mg/kg simmiparib, or vehicle (Veh) for 3 weeks. Data are depicted as mean ± SEM. *** P < 0.0001, ns, P = 0.7706. The IC 50 values of thioparib in six acquired PARPi‐resistant cell lines. Numbers 1–6 represent the PARPi‐resistant Capan‐1/OP, Capan‐1/TP, MDA‐MB‐436/OP, MDA‐MB‐436/TP, U251/OP, and U251/TP cells, respectively. Cells were treated with the indicated PARP inhibitors for 7 days and then subjected to SRB assay. The IC 50 values are expressed as the mean ± SD from three separate experiments. Efficacy of thioparib in the PARPi‐resistant model MDA‐MB‐436#AZD2281‐R1. Nude mice‐bearing MDA‐MB‐436#AZD2281‐R1 tumors ( n = 6) were orally dosed with 40 mg/kg thioparib and 100 mg/kg olaparib for 3 weeks. Mice tails show complete regression of tumors. Data are shown as mean ± SEM. *** P < 0.0001. Efficacy of thioparib in the PDX model BR‐05‐0028 (BRCA1‐deficient). Mice‐bearing BR‐05‐0028 tumors derived from a patient with breast cancer ( n = 6) were treated with 10 or 30 mg/kg thioparib or 100 mg/kg olaparib for 6 weeks. Data are depicted as mean ± SEM. *** P < 0.0001. Data information: Statistical analysis was performed by two‐way ANOVA. ThP, thioparib; OP, olaparib; TP, talazoparib; 391, Cpd‐391; SP, simmiparib.

Article Snippet: Antibody against BRCA1 (OP92) and BRCA2 (OP95) were from Millipore.

Techniques: CCK-8 Assay, Sulforhodamine B Assay, Derivative Assay