Journal: Journal of Medicinal Chemistry

Article Title: RTx-303, an Orally Bioavailable Polθ Polymerase Inhibitor That Potentiates PARP Inhibitors in BRCA Mutant Tumors

doi: 10.1021/acs.jmedchem.5c00551

Figure Lengend Snippet: Cellular and in vitro activity of RTx-284. A. Bar plot showing the IC50 of RTx-284 in the indicated HR-proficient (gray) and HRD (red) cell lines. Data represent mean of at least 3 independent experiments performed in triplicate. B-D. Synergy plots created by Combenefit showing synergistic activity between RTx-284 and indicated PARPi in the indicated HRD cell lines. E. Bar plot showing that 10 μM RTx-284 suppresses the MMEJ GFP reporter. Data represent mean of 3 individual experiments performed in triplicate ±SEM. F. Dot plot showing increase of γH2AX following RTx-284:olaparib treatment in DLD1 BRCA2–/– cells. G. Bar plot showing that RTx-284 promotes an increase in caspase positive DLD1 BRCA2–/– cells. Data represent mean of 3 independent experiments performed in triplicate, ±SEM.

Article Snippet: HCT 116 BRCA2 −/– and HCT 116 Parental were obtained from Cancertools, London, UK.

Techniques: In Vitro, Activity Assay

Journal: Journal of Medicinal Chemistry

Article Title: RTx-303, an Orally Bioavailable Polθ Polymerase Inhibitor That Potentiates PARP Inhibitors in BRCA Mutant Tumors

doi: 10.1021/acs.jmedchem.5c00551

Figure Lengend Snippet: RTx-303 potentiates PARPi in BRCA mutant cells and xenografts. A. Scatter plot showing relative viability of the indicated ID8 cells treated with the indicated concentrations of olaparib with and without 5 μM RTx-303. Data represent mean of 3 independent experiments performed in triplicate, ±SEM. B. Scatter plot showing BRCA2 mutant BR-05–0566 PDX volumes following treatment with vehicle, 45 mg/kg olaparib (PO,QD), 60 mg/kg RTx-303 (PO,BID), and 45 mg/kg olaparib (PO,QD) with 60 mg/kg RTx-303 (PO,BID) (left). Scatter plot showing % body weight change (right). Data represent mean, n = 7 ±SEM. C. Scatter plot showing BRCA2 mutant BR-05–0568 PDX volumes following treatment with vehicle, 0.12 mg/kg talazoparib (PO,QD) with and without 60 mg/kg RTx-303 (PO,BID) (left). Scatter plot showing % body weight change. (right). Data represent mean, n = 6 ±SEM. D. Scatter plot showing MDA-MB-436 tumor volumes following treatment with vehicle, 45 mg/kg olaparib (PO,QD) with and without 60 mg/kg RTx-303 (PO,BID) (left). Scatter plot showing % body weight change (right). Data represent mean, n = 8 ±SEM. * p < 0.05, *** p < 0.001.

Article Snippet: HCT 116 BRCA2 −/– and HCT 116 Parental were obtained from Cancertools, London, UK.

Techniques: Mutagenesis

Journal: Molecular medicine reports

Article Title: Effect of S100A8 and S100A9 on expressions of cytokine and skin barrier protein in human keratinocytes.

doi: 10.3892/mmr.2019.10454

Figure Lengend Snippet: Figure 6. HaCaT cells were treated with (A) S100A8 or (B) S100A9 (10 µg/ml) for 24 h and TLR4 expression was increased as assessed by western blotting. Data are expressed as the mean ± standard deviation. **P<0.01 vs. the control. S100, calcium binding protein; TLR4, toll‑like receptor 4.

Article Snippet: Tlr4 inhibitor cli-095 (Tlr4i), protein kinase δ (PKcδ) inhibitor (rottlerin), p38 mitogen-activated Correspondence to: dr in Sik Kim, department of Biomedical laboratory Science, School of Medicine, eulji university, 77 Gyeryoung-ro 771 beon-gil, Jung-Gu, daejeon 34824, republic of Korea e-mail: orientree@eulji.ac.kr Key words: atopic dermatitis, S100 calcium binding protein a8, S100 calcium binding protein a9, keratinocyte, skin barrier protein protein kinase (MaPK) inhibitor (SB202190), MeK inhibitor (Pd98059) and nuclear factor-κB (nF-κB) inhibitor (BaY-11-7085) were obtained from calbiochem (Merck KGaa). antibodies against p38 MaPK (cat. no. 9212), phospho-p38 MaPK (cat. no. 9211), phospho-extracellular signal regulated kinase 1/2 (erK1/2; cat. no. 9101), rabbit igG-HrP (cat. no. 7074), and mouse igG-HrP (cat. no. 7076) were acquired from cell Signaling Technology, inc. anti-erK2 (cat. no. sc-154) and anti-Tlr4 (cat. no. sc-10741) antibodies were obtained from Santa cruz Biotechnology, inc. Production of recombinant S100A8 and S100A9 proteins. in our previous report, the cdna of human S100a8 and S100a9 was cloned into peT28 expression vector (Merck KGaa) (10). recombinant S100a8 and S100a9 expression was induced with 1 mM isopropyl β-d-thiogalactoside in E. coli Bl21 (de3; Merck KGaa).

Techniques: Expressing, Western Blot, Standard Deviation, Control, Binding Assay

Journal: The Journal of cell biology

Article Title: RFWD3 promotes ZRANB3 recruitment to regulate the remodeling of stalled replication forks.

doi: 10.1083/jcb.202106022

Figure Lengend Snippet: Figure 1. Depletion of RFWD3 rescues HU sensitivity, nascent DNA degradation, and stalled fork collapse in BRCA2-deficient cells. (A) Detection of RFWD3 and BRCA2 levels in U2OS cells used in Fig. 1 B. (B) HU sensitivity of U2OS cells transfected with siBRCA2-3 and /or siRFWD3-4. Cell survival is normalized to the untreated control for each siRNA condition. Data represent the mean and SD of three replicates per HU dose and siRNA condition. Asterisks indicate P-values for RFWD3/BRCA2 versus BRCA2 depletion using an unpaired t test (*P < 0.05; ***P < 0.001; ****P < 0.0001). Data are representative of three independent experiments, for which mean LC50 values are provided in Fig. S1 B. (C) Schematic for single DNA fiber analysis to detect nascent DNA degradation at stalled forks. U2OS cells transfected with siBRCA2-3 and /or siRFWD3-4 were labeled with sequential CldU (25 min) and IdU (30 min) and then treated with 2 mM HU (5 h). Representative images are provided for replication tracks containing both CldU and IdU from cells transfected with the indicated siRNAs. Scale bars, 5 µm. (D) IdU/CldU replication track ratios in U2OS cells treated as in Fig. 1 C. Median values from >200 replication tracks are represented by red lines (n.s., not significant; ****P < 0.0001; Mann Whitney test). (E) Representative images of neutral comet tails in U2OS cells transfected with siBRCA2- 3 and/or siRFWD3-4 and treated with HU for 24 h. Scale bars, 50 µm. (F) Box plot of neutral comet-tail moments in U2OS cells from Fig. 1 E. Whiskers represent the 10th and 90th percentiles. More than 300 cells were scored for each condition (n.s., not significant; ****P < 0.0001; Mann Whitney test).

Article Snippet: For Western blot, the following rabbit antibodies were used: BRCA2 (A300-005A; Bethyl), FANCD2 (NB100-182; Novus), HLTF (ab183042; Abcam), PCNA (ab18197; Abcam), PCNA ubiquityl-Lys164 (13439; Cell Signaling), RFWD3 (A301-397A; Bethyl), RPA32 (A300-244A; Bethyl), RPA32 pS4/8 (A300-245A; Bethyl), RPA32 pT21 (ab109394; Abcam), RPA32 pS33 (A300246A; Bethyl, lot 3), SHPRH (ab80129; Abcam), β-Tubulin (2128; Cell Signaling), USP1 (A301-699A; Bethyl), and ZRANB3 (23111-1- AP; Proteintech).

Techniques: Transfection, Control, Labeling, MANN-WHITNEY

Journal: The Journal of cell biology

Article Title: RFWD3 promotes ZRANB3 recruitment to regulate the remodeling of stalled replication forks.

doi: 10.1083/jcb.202106022

Figure Lengend Snippet: Figure 2. Mutations in the RFWD3 ubiquitin ligase and WD40 domains rescue nascent DNA degradation in BRCA2-deficient cells. (A) Detection of RFWD3 and BRCA2 levels in U2OS cells for the experiment in Fig. 2 B. (B) U2OS cells expressing siRNA-resistant RFWD3 (WT or C315A) were transfected with siBRCA2-3 and siRFWD3-4, and they were compared with U2OS cells transfected with siBRCA2-3 with or without siRFWD3-4. Cells were labeled with se- quential CldU (25 min) and IdU (30 min) and then treated with 2 mM HU (5 h) as in Fig. 1 C. Median values for IdU/CldU track ratios (from >200 tracks) are represented by red lines (n.s., not significant; ****P < 0.0001; Mann Whitney test). (C) Detection of RFWD3 and BRCA2 levels in FA patient fibroblasts for the experiment in Fig. 2 D. (D) Schematic for single DNA fiber analysis to detect nascent DNA degradation at stalled forks in FA patient fibroblasts complemented with WT RFWD3 (1143 + WT) or empty vector (1143 + mock). Cells were labeled with sequential CldU (40 min) and IdU (50 min) and then treated with 2 mM HU (5 h). Representative images are provided for CldU and IdU-containing replication tracks upon transfection with siFF or siBRCA2-3. Scale bars, 5 µm. (E) IdU/ CldU replication length ratios in FA patient fibroblasts treated as in Fig. 2 D. Median values from >200 replication tracks are represented by red lines (n.s., not significant; ****P < 0.0001; Mann Whitney test).

Article Snippet: For Western blot, the following rabbit antibodies were used: BRCA2 (A300-005A; Bethyl), FANCD2 (NB100-182; Novus), HLTF (ab183042; Abcam), PCNA (ab18197; Abcam), PCNA ubiquityl-Lys164 (13439; Cell Signaling), RFWD3 (A301-397A; Bethyl), RPA32 (A300-244A; Bethyl), RPA32 pS4/8 (A300-245A; Bethyl), RPA32 pT21 (ab109394; Abcam), RPA32 pS33 (A300246A; Bethyl, lot 3), SHPRH (ab80129; Abcam), β-Tubulin (2128; Cell Signaling), USP1 (A301-699A; Bethyl), and ZRANB3 (23111-1- AP; Proteintech).

Techniques: Ubiquitin Proteomics, Expressing, Transfection, Labeling, MANN-WHITNEY, Plasmid Preparation

Journal: The Journal of cell biology

Article Title: RFWD3 promotes ZRANB3 recruitment to regulate the remodeling of stalled replication forks.

doi: 10.1083/jcb.202106022

Figure Lengend Snippet: Figure 6. RFWD3 stimulates ZRANB3 recruitment and replication fork reversal in BRCA2-deficient cells. (A) Immunoblot showing RFWD3, BRCA2, and HA-ZRANB3 levels in U2OS cells used in Fig. 6, B–E. HA-ZRANB3 was detected with anti-HA antibody. (B) U2OS cells expressing HA-ZRANB3 were transfected with siRFWD3-2 and/or siBRCA2-3, fixed 30 min after UV laser irradiation, and stained with anti-HA (green) and anti-γH2AX (red) antibodies. Scale bars, 10 µm. (C) Graph showing the percentage of γH2AX-positive cells with HA-ZRANB3 colocalization at UV laser stripes corresponding to the experiment in Fig. 6 B. Data represent the mean and SD from two independent experiments (**P < 0.01, unpaired t test). (D) U2OS cells expressing HA-ZRANB3 were transfected with siRFWD3-2 and/or siBRCA2-3 and treated with 10 µM EdU for 10 min followed by 1 µg/ml 4NQO for 4 h. After cell fixation, biotin was conjugated to EdU by click chemistry, and proximity ligation assay (PLA) was performed with anti-HA and anti-biotin antibodies. Images are representative of results quantitated in Fig. 6 E. Scale bars, 5 µm. (E) Box plot showing the distribution of PLA foci per cell for each condition (>200 cells) in Fig. 6 D. Whiskers represent the 10th and

Article Snippet: For Western blot, the following rabbit antibodies were used: BRCA2 (A300-005A; Bethyl), FANCD2 (NB100-182; Novus), HLTF (ab183042; Abcam), PCNA (ab18197; Abcam), PCNA ubiquityl-Lys164 (13439; Cell Signaling), RFWD3 (A301-397A; Bethyl), RPA32 (A300-244A; Bethyl), RPA32 pS4/8 (A300-245A; Bethyl), RPA32 pT21 (ab109394; Abcam), RPA32 pS33 (A300246A; Bethyl, lot 3), SHPRH (ab80129; Abcam), β-Tubulin (2128; Cell Signaling), USP1 (A301-699A; Bethyl), and ZRANB3 (23111-1- AP; Proteintech).

Techniques: Western Blot, Expressing, Transfection, Irradiation, Staining, Proximity Ligation Assay

Journal: The Journal of cell biology

Article Title: RFWD3 promotes ZRANB3 recruitment to regulate the remodeling of stalled replication forks.

doi: 10.1083/jcb.202106022

Figure Lengend Snippet: Figure 7. RFWD3 and ZRANB3 epistasis in replication fork remodeling phenotypes. (A) Detection of RFWD3, ZRANB3, and BRCA2 levels in U2OS cells used in Fig. 7, B and C. (B) U2OS cells were transfected with siBRCA2-3, siRFWD3-4, and/or siZRANB3. They were labeled with sequential CldU (25 min) and IdU (30 min) and then treated with 2 mM HU (5 h) as in Fig. 1 C. Median values for IdU/CldU track ratios (from >200 tracks) are represented by red lines (n.s., not significant; ****P < 0.0001; Mann Whitney test). (C) Neutral comet-tail moments in U2OS cells transfected with siBRCA2-3, siRFWD3-4, and/or siZRANB3 and treated with 2 mM HU for 24 h. Whiskers represent the 10th and 90th percentiles. More than 200 cells were analyzed for each condition (****P < 0.0001, Mann Whitney test). (D) Detection of RFWD3 and ZRANB3 levels in U2OS cells used in Fig. 7 E. (E) U2OS cells transfected with siRFWD3-4 and/or siZRANB3 were treated with 2 mM HU for 5 h. Replication intermediates were detected by electron microscopy, and the percentage of reversed forks was measured in a single replicate. The number of replication intermediates analyzed for each condition is indicated in parentheses.

Article Snippet: For Western blot, the following rabbit antibodies were used: BRCA2 (A300-005A; Bethyl), FANCD2 (NB100-182; Novus), HLTF (ab183042; Abcam), PCNA (ab18197; Abcam), PCNA ubiquityl-Lys164 (13439; Cell Signaling), RFWD3 (A301-397A; Bethyl), RPA32 (A300-244A; Bethyl), RPA32 pS4/8 (A300-245A; Bethyl), RPA32 pT21 (ab109394; Abcam), RPA32 pS33 (A300246A; Bethyl, lot 3), SHPRH (ab80129; Abcam), β-Tubulin (2128; Cell Signaling), USP1 (A301-699A; Bethyl), and ZRANB3 (23111-1- AP; Proteintech).

Techniques: Transfection, Labeling, MANN-WHITNEY, Electron Microscopy

Journal: PLoS ONE

Article Title: Establishment of primary mixed cell cultures from spontaneous canine mammary tumors: Characterization of classic and new cancer-associated molecules

doi: 10.1371/journal.pone.0184228

Figure Lengend Snippet: A . BRCA1 where * P < 0.05 versus NME, ** P < 0.05 versus CAd, *** P < 0.05 versus MAd, + P < 0.05 versus SCa, ++ P < 0.05 versus CCa1, +++ P < 0.05 versus CCa2. B . BRCA2 where * P < 0.05 versus NME, ** P < 0.05 versus CAd, *** P < 0.05 versus MAd, + P < 0.05 versus SCa, ++ P < 0.05 versus CCa1, +++ P < 0.05 versus CCa2. C . GATA3 where * P < 0.05 versus NME, ** P < 0.05 versus CAd, *** P < 0.05 versus MAd, + P < 0.05 versus SCa, ++ P < 0.05 versus CCa2. D . FGFR2 where * P < 0.05 versus NME, ** P < 0.05 versus CAd, *** P < 0.05 versus MAd, + P < 0.05 versus SCa, ++ P < 0.05 versus CCa1. Normal Mammary Epithelium (NME), Complex Adenoma (CAd), Mixed Adenoma (MAd), Simple Carcinoma (SCa), Complex Carcinoma 1 (CCa1), Complex Carcinoma 2 (CCa2), Mixed Carcinoma 1 (MCa1), Mixed Carcinoma 2 (MCa2).

Article Snippet: Assay numbers were as follows: FGFR2 (Cf02623903_m1, Cf02623899_m1), BRCA1 (Cf02625915_g1, Cf02625925_m1), MUC1 (Cf02626760_m1, Cf02680908_s1), BRCA2 (Cf02622077_m1, Cf02622063_m1), MYH11 (Cf02697091_m1, Cf02632489_m1), ESR1 (Cf02624844_m1, Cf02624844_m1), ESRRA (Cf02626582_gH), ESRRB (Cf02681395_s1, Cf02681401_s1), ESRRG (Cf00976241_m1, Cf00976243_m1) and B2M (Cf02659079_m1).

Techniques:

Journal: Cancer Biology & Therapy

Article Title: Clinical importance of FANCD2, BRIP1, BRCA1, BRCA2 and FANCF expression in ovarian carcinomas

doi: 10.1080/15384047.2019.1579955

Figure Lengend Snippet: Statistically significant associations of the BRCA2 and FANCD2 mRNA expression with overall survival (OS) in ovarian cancer patients, assessed in multivariate Cox proportional hazard models. Univariate analyses showed similar but weaker associations.

Article Snippet: Quantitative RT-PCR (Q-PCR) was run on the 7500 Fast Real-Time PCR System (Applied Biosystems), with the use of the FAM-labeled, TaqMan Gene Expression Assays (Applied Biosystems): FANCD2 (assay ID: Hs00276992_m1), BRIP1 (assay ID: Hs00230743_m1), BRCA1 (assay ID: Hs00173233_m1), BRCA2 (assay ID: Hs00609060_m1).

Techniques: Expressing

Journal: EMBO Reports

Article Title: Arginine methylation-dependent METTL14-SMN interaction regulates RNA m 6 A homeostasis

doi: 10.1038/s44319-025-00590-7

Figure Lengend Snippet: ( A ) Sanger sequencing confirmation of Mettl14 arginine methylation deficient mouse model. In total, 13 arginine resides located at the C-terminal disordered region of Mettl14 were mutated to lysine residues. ( B ) The arginine methylation levels of Mettl14 in WT, heterozygous (WT/RK), and homozygous (RK/RK) mice were detected by IP-western blot using tissue lysates from mouse spleen and thymus. ( C ) The protein expression levels of several DNA repair genes, including BRCA1, ATRIP, and PALB2, were detected by western blot using Thymus tissues from WT, WT/RK, and RK/RK mice. ( D ) Mettl14 arginine methylation deficiency increases the population of primitive hematopoietic cells (LK cells). ** P = 0.0019. ( E ) Mettl14 arginine methylation deficiency increases the population of myeloid (Mac1 + Gr1 + ) cells in the BM, but not in the spleen. * P = 0.013. ( F ) Mettl14 arginine methylation deficiency decreases the B lymphoid (B220 + ) population in the BM, but not in the spleen. * P = 0.011. ( G ) Mettl14 arginine methylation deficiency decreases the T lymphoid (CD3 + ) population in the BM, but not in the spleen. Data from three independent replicates were analyzed by Student’s t test and shown as mean ± SD. ** P = 0.0097. .

Article Snippet: Rabbit anti-PALB2 antibody , Proteintech , 14340-1-AP.

Techniques: Sequencing, Methylation, Western Blot, Expressing

Journal: bioRxiv

Article Title: Loss of E2F7 confers resistance to poly-ADP-ribose polymerase (PARP) inhibitors in BRCA2-deficient cells

doi: 10.1101/294371

Figure Lengend Snippet: A . Western blot showing loss of BRCA2 expression in HeLa cells with CRISPR/Cas9-mediated BRCA2 knockout. B . BRCA2 KO HeLa cells have similar olaparib sensitivity as HeLa cells treated with BRCA2 siRNA. Results are shown as normalized to control (no drug treatment) for each sample. The average of three experiments, with error bars as standard deviations, is shown. C, D . E2F7 knockdown rescues the olaparib sensitivity of BRCA2-knockout ( C ) and BRCA2-knockdown ( D ) HeLa cells. The average of three experiments, with error bars as standard deviations, is shown. E . Quantitative RT-PCR experiment showing efficient E2F7 knockdown by the siRNA oligonucleotides employed. HeLa cells were treated with the indicated siRNA oligonucleotides, than incubated with 10μM olaparib for 24h before harvesting. The average of three experiments, with error bars as standard deviations, is shown. F . Western blot showing that BRCA2 is efficiently knocked down by the siRNA oligonucleotide employed singly or in combination with E2F7 siRNA oligonucleotides. HeLa cells were treated with the indicated siRNA oligonucleotides, than incubated with 10μM olaparib for 24h before harvesting. G . Quantification of AnnexinV-positive cells indicating that E2F7 knockdown suppresses olaparib-induced apoptosis of BRCA2-depleted cells. HeLa cells were treated with the indicated siRNA oligonucleotides, than incubated with 5μM olaparib for 3 days. Results are presented as normalized to control (no drug treatment condition) for each knockdown sample. The average of three independent experiments, with standard deviations as error bars, is shown.

Article Snippet: For BRCA2 gene knockout, the commercially available BRCA2 CRISPR/Cas9 KO plasmid was used (Santa Cruz Biotechnology sc-400700).

Techniques: Western Blot, Expressing, CRISPR, Knock-Out, Control, Knockdown, Quantitative RT-PCR, Incubation

Journal: bioRxiv

Article Title: Loss of E2F7 confers resistance to poly-ADP-ribose polymerase (PARP) inhibitors in BRCA2-deficient cells

doi: 10.1101/294371

Figure Lengend Snippet: A, B . E2F7 knockdown rescues the olaparib sensitivity of BRCA2-knockdown HCC1395 breast cancer ( A ) and SH-SY5Y neuroblastoma ( B ) cells. HCC1395 cells are BRCA1-mutant, which accounts for their increased olaparib sensitivity. The average of three experiments, with error bars as standard deviations, is shown. C . E2F7 depletion increases resistance of SH-SY5Y cells to olaparib-camptothecin combination treatment. The average of three technical replicates, with error bars as standard deviations, is shown. D . E2F7 knockdown rescues the cisplatin sensitivity of BRCA2-knockdown HeLa cells. The average of three experiments, with error bars as standard deviations, is shown.

Article Snippet: For BRCA2 gene knockout, the commercially available BRCA2 CRISPR/Cas9 KO plasmid was used (Santa Cruz Biotechnology sc-400700).

Techniques: Knockdown, Mutagenesis

Journal: bioRxiv

Article Title: Loss of E2F7 confers resistance to poly-ADP-ribose polymerase (PARP) inhibitors in BRCA2-deficient cells

doi: 10.1101/294371

Figure Lengend Snippet: A . Quantitative RT-PCR experiment showing increased RAD51 mRNA levels upon E2F7 depletion in BRCA2-knockdown HeLa cells incubated with 10μM olaparib for 24h before harvesting. The average of three experiments, with error bars as standard deviations, is shown. Asterisks denote statistical significance compared to siBRCA2 condition (using the two-tailed equal variance TTEST). B . Western blot showing that E2F7 knockdown results in increased RAD51 protein levels in HeLa cells treated with 10μM olaparib for 24h. C . Chromatin fractionation experiment showing that E2F7 depletion results in increased chromatin-bound RAD51. HeLa cells were treated with 10μM olaparib for 24h. Vinculin is used as loading control. D . DR-GFP assay in U2OS cells showing that E2F7 depletion restores HR levels in BRCA2-knockdown cells, but not in RAD51-knockdown cells. The average of three experiments, with error bars as standard deviations, is shown. Asterisks indicate statistical significance (using the two-tailed equal variance TTEST).

Article Snippet: For BRCA2 gene knockout, the commercially available BRCA2 CRISPR/Cas9 KO plasmid was used (Santa Cruz Biotechnology sc-400700).

Techniques: Quantitative RT-PCR, Knockdown, Incubation, Two Tailed Test, Western Blot, Fractionation, Control

Journal: bioRxiv

Article Title: Loss of E2F7 confers resistance to poly-ADP-ribose polymerase (PARP) inhibitors in BRCA2-deficient cells

doi: 10.1101/294371

Figure Lengend Snippet: A . Schematic representation of the experimental setup for the DNA fiber combing experiment. B . Quantification of the IdU tract length. BRCA2 knockdown reduces the length of the IdU tract upon HU treatment, indicating that the nascent strand is degraded. This degradation can be suppressed by E2F7 depletion, or as control, by incubation with the MRE11 inhibitor mirin. The median is indicated.

Article Snippet: For BRCA2 gene knockout, the commercially available BRCA2 CRISPR/Cas9 KO plasmid was used (Santa Cruz Biotechnology sc-400700).

Techniques: Knockdown, Control, Incubation