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: 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: 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: Oncology Letters

Article Title: Olaparib promotes FABP4 expression and reduces antitumor effect in ovarian cancer cells with a BRCA1 mutation

doi: 10.3892/ol.2024.14813

Figure Lengend Snippet: AZD2281 upregulates FABP4 expression. (A) mRNA expression of BRCA1, BRCA2, PPARγ and FABP4 at different time points (ctrl, 12, 24 and 48 h) after treatment with 400 nM AZD2281 in OVCAR5 and COV362 cells. ***P<0.001. (B) Protein expression of BRCA1, BRCA2, PPARγ, cEBPα and FABP4 at different time points (ctrl, 12, 24 and 48 h) after treatment with 400 nM AZD2281 in OVCAR5 and COV362 cells. *P<0.05 vs. Ctrl;**P<0.01 vs. Ctrl;***P<0.001 vs. Ctrl; ****P<0.0001 vs. Ctrl. Fold enrichments of cEBPα in the (C) FABP4 and (D) PPARγ promoters at different time points (ctrl, 12, 24 and 48 h) after treatment with 400 nM AZD2281. *P<0.05 vs. Ctrl; **P<0.01 vs. Ctrl; ***P<0.001 vs. Ctrl; ***P<0.001 vs. Ctrl. FABP4, fatty acid binding protein 4; BRCA, breast cancer susceptibility gene; PPARγ, peroxisome proliferator activated receptor γ; cEBPα, CCAAT enhancer binding protein α; ChIP, chromatin immunoprecipitation; Ctrl, control.

Article Snippet: The membranes were incubated at 4°C overnight with the following antibodies: Anti-FABP4 (1:1,000; cat. no. ab92501; Abcam), anti-Snail (1:1,000; cat. no. 3879T; CST Biological Reagents Co., Ltd.), anti-Slug (1:1,000; cat. no. ab27568; Abcam), anti-B-cell lymphoma 2 (BCL2; 1:1,000; cat. no. AB1722; MilliporeSigma), anti- BRCA1 (1:1,000; cat. no. 22362-1-AP; Proteintech Group, Inc.), anti- BRCA2 (1:1,000; cat. no. 29450-1-AP; Proteintech Group, Inc.), anti-cEBPα (1:1,000; cat. no. 18311-1-AP; Proteintech Group, Inc.), anti-peroxisome proliferator activated receptor γ (PPARγ; 1:1,000; cat. no. 16643-1-AP; Proteintech Group, Inc.) and anti-GAPDH (1:20,000; cat. no. 5174; CST Biological Reagents Co., Ltd.).

Techniques: Expressing, Binding Assay, Chromatin Immunoprecipitation, Control

Journal: Gynecologic Oncology Reports

Article Title: BRCA immunohistochemistry for screening of BRCA mutation in epithelial ovarian cancer patients

doi: 10.1016/j.gore.2020.100582

Figure Lengend Snippet: Performance of BRCA1 immunohistochemistry (IHC) and BRCA mutation status.

Article Snippet: Samples were stained and incubated for 60 min with BRCA1 mouse monoclonal antibody (Novus biological Inc., USA) and BRCA2 rabbit polyclonal antibody (Novus biological Inc., USA) at a dilution ratio of 1:100.

Techniques: Immunohistochemistry, Mutagenesis

Journal: Gynecologic Oncology Reports

Article Title: BRCA immunohistochemistry for screening of BRCA mutation in epithelial ovarian cancer patients

doi: 10.1016/j.gore.2020.100582

Figure Lengend Snippet: Performance of BRCA2 immunohistochemistry (IHC) and BRCA mutation status.

Article Snippet: Samples were stained and incubated for 60 min with BRCA1 mouse monoclonal antibody (Novus biological Inc., USA) and BRCA2 rabbit polyclonal antibody (Novus biological Inc., USA) at a dilution ratio of 1:100.

Techniques: Immunohistochemistry, Mutagenesis

Journal: Gynecologic Oncology Reports

Article Title: BRCA immunohistochemistry for screening of BRCA mutation in epithelial ovarian cancer patients

doi: 10.1016/j.gore.2020.100582

Figure Lengend Snippet: Performance of BRCA 1/2 immunohistochemistry (IHC) for detection of germline BRCA1/2 mutation.

Article Snippet: Samples were stained and incubated for 60 min with BRCA1 mouse monoclonal antibody (Novus biological Inc., USA) and BRCA2 rabbit polyclonal antibody (Novus biological Inc., USA) at a dilution ratio of 1:100.

Techniques: Immunohistochemistry, Mutagenesis

Journal: Med (New York, N.Y.)

Article Title: Inactivation of TACC2 epigenetically represses CDKN1A and confers sensitivity to CDK inhibitors.

doi: 10.1016/j.medj.2024.12.002

Figure Lengend Snippet: Figure 3. TACC2 interferes with the NuRD and CoREST corepressor complexes (A) SDS-PAGE and silver staining analysis of proteins immunoprecipitated using an anti-GFP antibody from EGFP-TACC2- or EGFP-overexpressing cells (left). The major interacting proteins are indicated on the right. (B) Schematic of N-terminal EGFP-tagged full-length TACC2 (FL) and the indicated deletion mutants (top) and IP coupled with immunoblot analysis of HEK293T cells transfected with the indicated constructs using the indicated antibodies (bottom). (C) IP of the endogenous TACC2 in ECA109 and KYSE30 cells followed by WB assays using the indicated proteins. (D) HEK293T cells were cotransfected with 3 mg each of vectors expressing FLAG-MTA1 and Myc-MBD3 or expressing FLAG-HMG20B and Myc-LSD1 together with 0, 3, and 6 mg of the vector expressing EGFP-TACC2 for 36 h. IP and WB were then performed with the indicated antibodies. (E) ECA109 cells were transfected with two TACC2 siRNAs and siNC for 48 h. IP and WB were performed with the indicated antibodies. (F) Representative immunofluorescence images of endogenous TACC2, MTA1, MBD3, or HMG20B in ECA109 cells. Green, TACC2; red, MTA1, MBD3, and HMG20B; blue, nuclei. Scale bars, 20 mm. (G) Representative immunofluorescence images of ECA109 cells transfected with TACC2 siRNAs or siNC for 48 h and stained with the indicated antibodies (left) and quantification of the nuclear localization percentage of MTA1, MBD3, or HMG20B (right). Scale bars, 20 mm. At least 100 cells were analyzed. Data are the mean ± SD of 3 independent experiments. Significance was measured by one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001. (H) Cytoplasmic and nuclear proteins from ECA109 cells transfected with TACC2 siRNAs or siNC for 48 h were separated and subjected to WB. (I) IHC analyses were performed with the indicated antibodies on serial sections of ESCC specimens (n = 45). Representative images are shown (left). Scale bars, 200 mm. Correlation between TACC2 levels and the cytoplasm localization of indicated proteins was determined (right). Two-tailed chi-square test; ***p < 0.001. See also Figure S5 and Table S3.

Article Snippet: After the retrieval solution had cooled to room temperature, sections were incubated with diluted anti-TACC2 antibody (rabbit polyclonal, 1:200; 11407-1-AP; Proteintech), anti-Ki67 antibody (rabbit polyclonal, 1:1000; 27309-1-AP; Proteintech), anti-Ki67 antibody (rabbit monoclonal, 1:200; #12202; CST), antiCD3 antibody (rabbit monoclonal, 1:100; ab5690; Abcam), anti-CD11b antibody (rabbit monoclonal, 1:100; ab133357; Abcam), anti-p21 antibody (rabbit monoclonal, 1:200; #2947; CST), anti-MTA1 antibody (rabbit monoclonal, 1:200; #5646; CST), antiMBD3 antibody (rabbit polyclonal, 1:200; 14258-1-AP; Proteintech), anti-HMG20B antibody (rabbit polyclonal, 1:200; 14582-1- AP; Proteintech), overnight at 4 C. Sections were then washed three times with phosphate-buffered saline containing Tween 20 (PBST; ZSGB-BIO) and incubated with goat anti-rabbit peroxidase-conjugated secondary antibodies (DAKO, Santa Clara) for 30min at 37 C. Sections were thenwashed three timeswith PBST and stainedwith 3,30-diaminobenzidine (DAB) for 2min to visualize the target protein.

Techniques: SDS Page, Silver Staining, Immunoprecipitation, Western Blot, Transfection, Construct, Expressing, Plasmid Preparation, Staining, Two Tailed Test