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: 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: Journal of Clinical Oncology

Article Title: Detecting BRCA2 Protein Truncation in Tissue Biopsies to Identify Breast Cancers That Arise in BRCA2 Gene Mutation Carriers

doi: 10.1200/JCO.2008.20.5211

Figure Lengend Snippet: Dual C-terminal and N-terminal BRCA2 immunohistochemistry (IHC) of hereditary breast cancers (A and B, respectively) and of sporadic breast cancers (C and D, respectively). Magnification is (left) ×20 and (right) ×100 for all panels. The upper panel for each pair is C-terminal IHC, and the lower panel is N-terminal IHC. (A) Samples are from a BRCA2-mutant cancer with 7231del5. Top panel stained with C-terminal BRCA2 antibody, and second panel stained with N-terminal BRCA2 antibody. Only a lymphocyte in the top panel stains. (B) Samples are from a patient with a 9654delTT BRCA2 mutation at similar magnifications and similar staining. (C and D) The paired panels are from adjacent sections of the same sporadic breast cancer sample. Note the similar nuclear staining with both antibodies for the sporadic cancer samples.

Article Snippet: The N-terminal antibody used was from R&D systems MAB2476 (a commercially available mouse monoclonal antibody directed against an E coli –derived recombinant human BRCA2 protein that spans amino acids 1-200), which was used at 3 μg/mL.

Techniques: Immunohistochemistry, Mutagenesis, Staining

Journal: Journal of Clinical Oncology

Article Title: Detecting BRCA2 Protein Truncation in Tissue Biopsies to Identify Breast Cancers That Arise in BRCA2 Gene Mutation Carriers

doi: 10.1200/JCO.2008.20.5211

Figure Lengend Snippet: Immunohistochemistry with 575A15 C-terminal monoclonal antibody on normal breast epithelial lobules. Upper panels: untreated antibody. Middle panels: antibody mixed with excess of immunizing peptide (BRCA2 amino acids 3284 to 3294: TFVSPAAKAGG). Lower panels: antibody mixed with excess of control BRCA2 peptide (BRCA2 amino acids between 3300 and 3400, obtained from Abcam (Cambridge MA). Magnification is (left) ×20 and (right) ×100.

Article Snippet: The N-terminal antibody used was from R&D systems MAB2476 (a commercially available mouse monoclonal antibody directed against an E coli –derived recombinant human BRCA2 protein that spans amino acids 1-200), which was used at 3 μg/mL.

Techniques: Immunohistochemistry

Journal: Journal of Clinical Oncology

Article Title: Detecting BRCA2 Protein Truncation in Tissue Biopsies to Identify Breast Cancers That Arise in BRCA2 Gene Mutation Carriers

doi: 10.1200/JCO.2008.20.5211

Figure Lengend Snippet: MCF7 cells were cultured for 48 hours in 10% charcoal-stripped serum/phenol red–free DMEM and were treated with 10 nmol/L estrogen for 5 and 30 minutes and for 1, 2, 4, and 24 hours. Samples were blotted with the 575A15 BRCA2 C-terminal monoclonal antibody. The 440 kDa band is the only band on the blot.

Article Snippet: The N-terminal antibody used was from R&D systems MAB2476 (a commercially available mouse monoclonal antibody directed against an E coli –derived recombinant human BRCA2 protein that spans amino acids 1-200), which was used at 3 μg/mL.

Techniques: Cell Culture

Journal: Journal of Clinical Oncology

Article Title: Detecting BRCA2 Protein Truncation in Tissue Biopsies to Identify Breast Cancers That Arise in BRCA2 Gene Mutation Carriers

doi: 10.1200/JCO.2008.20.5211

Figure Lengend Snippet: BRCA2 Mutations Identified in Patients With Breast Cancer

Article Snippet: The N-terminal antibody used was from R&D systems MAB2476 (a commercially available mouse monoclonal antibody directed against an E coli –derived recombinant human BRCA2 protein that spans amino acids 1-200), which was used at 3 μg/mL.

Techniques: 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: Fig. 4 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: Frontiers in Cell and Developmental Biology

Article Title: Mesenchymal Stem Cells Early Response to Low-Dose Ionizing Radiation

doi: 10.3389/fcell.2020.584497

Figure Lengend Snippet: LDIR affect the processes of apoptosis and repair in the cells. (A) Quantitative reverse transcriptase–polymerase chain reaction. (1) and (2) Changes in the levels of RNA BRCA1 and RNA BRCA2 in LDIR-treated cells compared to control (three biological replicates). The time of cell cultivation after LDIR is shown in the figure. (3) Proapoptotic gene BAX 1 RNA level and BIRC and BCL families antiapoptotic genes changes in the MSCs (10 cGy) compared to control (three biological replicates). Reference gene TBP . * p < 0.001 ( U test). (B) FACS. BRCA2 protein level changes. (1) Plots: FL1- BRCA2 versus SSC. R: gated area, cells with an increased BRCA2 level. (2) MSCs (10 cGy, 15 min after LDIR) with varying FL1–BRCA2 signal distribution. (3) The content of the cells with an increased BRCA2 level. (4) Median FL1–BRCA2 signals. Average values and SD are given for three experiments. * p < 0.001 ( U test). The time of cell cultivation after LDIR is shown in the figure. (C) FACS. (1) The cumulative distribution of the MSCs (10 and 50 cGy) with varying FL1–BAX1 signal. (2) Median signals for FL1–BAX1. (3) The cumulative distribution of the MSCs (10 and 50 cGy) with varying FL1–BCL2 signal. (4). Median signals for FL1–BCL2. The cultivation times and radiation doses are shown in the figure. Average values and SD are given for three experiments. * p < 0.001 ( U test). (D) FACS. (1–3) The cumulative distribution of the MSCs (10 cGy) with varying FL1–p53, FL1-ATM, and FL1–PPARG signals. The time of cell cultivation after LDIR is shown in the figure. (E) FACS (1, 2). (1) The distribution of the MSCs (50 cGy) with varying FL1–annexin V. R: gated area, cells with an increased annexin V level. (2) The content of the cells with an increased annexin V level. (3) Evaluation of cell viability 72 h after irradiation. MTT test, the number of the attached cells, and the total cell number. Parameter values are normalized to the control values.

Article Snippet: The following antibodies were used: γ H2AX- Dylight488 (pSer139) (NB100-78356G, Novus Biologicals); 8OHDG (Sc-66036, Santa Cruz Biotechnology); NOX4 (Sc-30141, Santa Cruz Biotechnology); NRF2 (ab137550); pNRF2 (Ser40) (Bioss Inc; BS-2013R), BRCA2 (NBP1-88361, Novus Biologicals); proliferating cell nuclear antigen (PCNA) (ab2426, Abcam); ATM; P53; PPARG; KI-67 fluorescein isothiocyanate (FITC) (sc-23900 FITC) (Santa Cruz Biotechnology); and BCL2 (Sc-783, Santa Cruz Biotechnology).

Techniques: Reverse Transcription, Polymerase Chain Reaction, Control, Irradiation