Journal: Cell reports

Article Title: NEIL3 contributes to the Fanconi anemia/BRCA pathway by promoting the downstream double-strand break repair step.

doi: 10.1016/j.celrep.2022.111600

Figure Lengend Snippet: Figure 4. NEIL3 is required for DSB end resection (A–D) Representative images and statistical analysis to show (A, B) RPA2 foci and (C, D) BrdU foci formation 24 h after 100 ng/mL MMC treatment in WT and NEIL3/ HeLa cells. Scale bar, 10 mm. A total of 100 cells from three independent experiments were analyzed for each group. ***p < 0.001, ****p < 0.0001, two- tailed Student’s t test. (E) The schematic of in vitro DSB end resection assay in ER-AsiSI U2OS cells. See STAR Methods for details. (F) Quantification and statistical analysis of the ssDNA level after depletion of NEIL3, CtIP, and BRCA1, respectively, in ER-AsiSI U2OS cells. Data are shown as mean ± SEM compiled from three independent experiments. **p < 0.01, ***p < 0.001, two-tailed Student’s t test. (G) The efficiency of knockdown of individual proteins was examined by western blot.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 4-Hydroxytamoxifen Sigma-Aldrich Cat# H7904 Ethidium Bromide Sigma-Aldrich Cat# E8751 Doxycycline Selleck Chemicals Cat# S5159 Hoechst 33,342 Beyotime Technology Cat# C1029 Doxycycline Selleck Chemicals Cat# S5159 Benzonase Sigma-Aldrich Cat# E8263 MG-132 Selleck Chemicals Cat# S2619 Olaparib (AZD2281) Selleck Chemicals Cat# S1060 Critical commercial assays CellTiter-Glo Luminescent Viability Assay Promega Cat# G7570 Subcellular Protein Fractionation Kit Life Technologies Cat# 78840 CometAssay Single Cell Gel Electrophoresis Assay Trevigen Cat# 4250-050-K Hieff Clone Plus One Step Cloning Kit Yeasen Cat# 10911ES20 Immunoprecipitation (IP/CoIP) Kit Absin Cat# abs955 TIANamp Genomic DNA Kit TIANGEN Cat# DP304 PierceTM anti-HA magnetic beads Thermo Fisher Scientific Cat# 88836 PierceTM anti-c-Myc magnetic beads Thermo Fisher Scientific Cat# 88842 GFP-Trap magnetic beads Chromotek Cat# gtma-20 Experimental models: Cell lines 293T Cell Line ATCC N/A HeLa Cell Line ATCC N/A U2OS Cell Line ATCC N/A RPE1 Cell Line ATCC N/A U2OS-HA-ER-AsiSI Cell Line Gift from Dr. Gaëlle Legube N/A U2OS-FANCD2 / Cell Line Li et al., 2020 N/A HeLa-FANCA / Cell Line Li et al., 2020 N/A HeLa-NEIL3 / Cell Line Li et al., 2020 N/A HeLa-NEIL3-WT-Dox Cell line This paper N/A HeLa-NEIL3-del N term-Dox Cell line This paper N/A HeLa-NEIL3-del C term-Dox Cell line This paper N/A HeLa-NEIL3-del GRF2 -Dox Cell line This paper N/A HeLa-NEIL3-del GRF 1-2-Dox Cell line This paper N/A Oligonucleotides siRNA targeting sequence: negative Control: GGGTATCGACGATTACAAA Li et al., 2020 N/A siRNA targeting sequence: NEIL3 #1: GGGTGGATCATGTTATGGA Li et al., 2020 N/A siRNA targeting sequence: NEIL3 #2: GCTAATGGATCAGAACGTA Li et al., 2020 N/A siRNA targeting sequence: NEIL3 #50UTR: TGAGTTGCACAGCGGTATT This paper N/A siRNA targeting sequence: FANCA: TTTTTCCCTCTTGACCCTT Li et al., 2020 N/A siRNA targeting sequence: FANCD2: GGCTCAGGATTCTAATGTA Li et al., 2020 N/A siRNA targeting sequence: NBS1: CCAACTAAATTGCCAAGTA Sangon Biotech N/A (Continued on next page) e2 Cell Reports 41, 111600, November 8, 2022

Techniques: Two Tailed Test, In Vitro, Resection Assay, Knockdown, Western Blot

Journal: Cell reports

Article Title: NEIL3 contributes to the Fanconi anemia/BRCA pathway by promoting the downstream double-strand break repair step.

doi: 10.1016/j.celrep.2022.111600

Figure Lengend Snippet: Figure 6. NEIL3 acts upstream of CtIP and MRN complex to promote their recruitment to DSBs (A) Western blots to validate the expression of GFP-tagged constructs of NEIL3, CtIP, and NBS1, respectively. (B) Representative time-lapse view to show the recruitment of indicated proteins to laser irradiation damaged sites in U2OS cells. Scale bar, 5 mm. (C) Normalized fluorescent intensity curve of indicated proteins at DNA damage sites. (legend continued on next page)

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 4-Hydroxytamoxifen Sigma-Aldrich Cat# H7904 Ethidium Bromide Sigma-Aldrich Cat# E8751 Doxycycline Selleck Chemicals Cat# S5159 Hoechst 33,342 Beyotime Technology Cat# C1029 Doxycycline Selleck Chemicals Cat# S5159 Benzonase Sigma-Aldrich Cat# E8263 MG-132 Selleck Chemicals Cat# S2619 Olaparib (AZD2281) Selleck Chemicals Cat# S1060 Critical commercial assays CellTiter-Glo Luminescent Viability Assay Promega Cat# G7570 Subcellular Protein Fractionation Kit Life Technologies Cat# 78840 CometAssay Single Cell Gel Electrophoresis Assay Trevigen Cat# 4250-050-K Hieff Clone Plus One Step Cloning Kit Yeasen Cat# 10911ES20 Immunoprecipitation (IP/CoIP) Kit Absin Cat# abs955 TIANamp Genomic DNA Kit TIANGEN Cat# DP304 PierceTM anti-HA magnetic beads Thermo Fisher Scientific Cat# 88836 PierceTM anti-c-Myc magnetic beads Thermo Fisher Scientific Cat# 88842 GFP-Trap magnetic beads Chromotek Cat# gtma-20 Experimental models: Cell lines 293T Cell Line ATCC N/A HeLa Cell Line ATCC N/A U2OS Cell Line ATCC N/A RPE1 Cell Line ATCC N/A U2OS-HA-ER-AsiSI Cell Line Gift from Dr. Gaëlle Legube N/A U2OS-FANCD2 / Cell Line Li et al., 2020 N/A HeLa-FANCA / Cell Line Li et al., 2020 N/A HeLa-NEIL3 / Cell Line Li et al., 2020 N/A HeLa-NEIL3-WT-Dox Cell line This paper N/A HeLa-NEIL3-del N term-Dox Cell line This paper N/A HeLa-NEIL3-del C term-Dox Cell line This paper N/A HeLa-NEIL3-del GRF2 -Dox Cell line This paper N/A HeLa-NEIL3-del GRF 1-2-Dox Cell line This paper N/A Oligonucleotides siRNA targeting sequence: negative Control: GGGTATCGACGATTACAAA Li et al., 2020 N/A siRNA targeting sequence: NEIL3 #1: GGGTGGATCATGTTATGGA Li et al., 2020 N/A siRNA targeting sequence: NEIL3 #2: GCTAATGGATCAGAACGTA Li et al., 2020 N/A siRNA targeting sequence: NEIL3 #50UTR: TGAGTTGCACAGCGGTATT This paper N/A siRNA targeting sequence: FANCA: TTTTTCCCTCTTGACCCTT Li et al., 2020 N/A siRNA targeting sequence: FANCD2: GGCTCAGGATTCTAATGTA Li et al., 2020 N/A siRNA targeting sequence: NBS1: CCAACTAAATTGCCAAGTA Sangon Biotech N/A (Continued on next page) e2 Cell Reports 41, 111600, November 8, 2022

Techniques: Western Blot, Expressing, Construct, Irradiation

Journal: Oncogene

Article Title: BRCA1 physically associates with p53 and stimulates its transcriptional activity.

doi: 10.1038/sj.onc.1201932

Figure Lengend Snippet: Figure 2 BRCA1 interacts with p53 both in vitro and in vivo. (a) GST or GST-p53 proteins were expressed in E. coli and mixed with in vitro-translated (IVT) 35S-methionine-labeled BRCA1 (lanes 2, 5 and 6) or luciferase (negative control; lanes 1, 3 and 4), precipitated with Glutathione-sepharose beads, and analysed by 7.5% SDS ± PAGE. In lanes 1 and 2, 1/10th of the IVT-labeled proteins used for binding assays were loaded directly. (b) GST-p53 interacts with endogenous BRCA1. GST-p53 expressed as above was mixed with COS-7 cell lysate, precipitated with Glutathione-sepharose beads, separated by 7.5% SDS ± PAGE, and analysed by immunoblotting with anti-BRCA1 (SG11) antibody. (c) Total cell lysate from COS-7 (lane 1), HeLa (lane 2) and U2OS (lane 3) cells were analysed by Western blotting for BRCA1 with anti-BRCA1 (SG11) antibody. (d) Total cell lysate from COS-7 (lane 1), HeLa (lane 2) and U2OS (lane 3) cells were immunoprecipitated with anti-BRCA1 (C-20) and the immunoprecipitates were analysed by anti-BRCA1 (SG11). (e) COS-7 extracts were immunoprecipitated with anti-p16 (lane 2; negative control), anti-BRCA1 antibodies, C20 (lane 3) and SG11 (lane 4), anti-MDM-2 (Ab1) antibody (lane 5: positive control) and anti-p53 (lane 6). (f) HeLa cell extracts were immunoprecipitated with anti-p16 (lane 1), anti-BRCA1 antibodies C20 (lane 2), SG11 (lane 3) and anti-p53 (lane 4). (g) U2OS cell extracts were immunoprecipitated with anti-p16 (lane 2; negative control), anti-BRCA1 antibodies, SG11 (lane 3), anti-MDM-2 antibody (lane 4; positive control) and anti-p53, Ab6 (lane 6). The co-precipitated p53 (Figure 2e, f and g) was detected by immunoblotting with HRP-conjugated anti-p53 antibody as described in Materials and methods. (h) SW480 cell extracts were immunoprecipitated with anti-BRCA1 antibody, Ab1 (lane 1) and IgG (lane 2; negative control). The co-precipitated p53 was detected by immunoblotting with anti-p53 (Ab7) antibody and anti-mouse HRP antibody

Article Snippet: SAOS-2, HeLa, COS7, HBL100, U2OS and 293 cells were obtained from ATCC.

Techniques: In Vitro, In Vivo, Labeling, Luciferase, Negative Control, SDS Page, Binding Assay, Western Blot, Immunoprecipitation, Positive Control

Journal: Molecular medicine reports

Article Title: Downregulation of 14-3-3β inhibits proliferation and migration in osteosarcoma cells.

doi: 10.3892/mmr.2017.8144

Figure Lengend Snippet: Figure 1. Endogenous 14‑3‑3β expression in osteosarcoma tissues and in matched normal tumor‑adjacent bone tissues from patients. (A) 14‑3‑3β mRNA expression levels were assessed by reverse transcription‑quantitative polymerase chain reaction. *P<0.05. (B) 14‑3‑3β protein expression levels were assessed by western blotting. All data are presented as mean ± standard deviation.

Article Snippet: Human osteoblastic cell line (hFOB1.19) and osteosarcoma cell lines (U2OS, Saos-2 and MG63) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Polymerase Chain Reaction, Western Blot, Standard Deviation

Journal: Molecular medicine reports

Article Title: Downregulation of 14-3-3β inhibits proliferation and migration in osteosarcoma cells.

doi: 10.3892/mmr.2017.8144

Figure Lengend Snippet: Figure 2. Endogenous 14‑3‑3β expression in osteosarcoma cell lines. Endogenous expression of 14‑3‑3β was assessed in the osteosarcoma cell lines U2OS, Saos‑2 and MG63 and in the human normal osteoblast cell line hFOB1.19. (A) 14‑3‑3β mRNA expression levels were assessed by reverse transcription‑quantitative polymerase chain reaction. (B) 14‑3‑3β protein expression levels were assessed by western blotting. (C) Quantification of protein expression relative to β‑actin (loading control). All data are presented as mean ± standard deviation. *P<0.05 vs. hFOB1.9.

Article Snippet: Human osteoblastic cell line (hFOB1.19) and osteosarcoma cell lines (U2OS, Saos-2 and MG63) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Polymerase Chain Reaction, Western Blot, Control, Standard Deviation

Journal: Molecular medicine reports

Article Title: Downregulation of 14-3-3β inhibits proliferation and migration in osteosarcoma cells.

doi: 10.3892/mmr.2017.8144

Figure Lengend Snippet: Figure 3. 14‑3‑3β knockdown in MG63 osteosarcoma cells. MG63 cells were transfected with either a 14‑3‑3β‑specific siRNA (knockdown group) or a non‑specific control siRNA (control group). Untransfected cells were used as the blank group. (A) 14‑3‑3β mRNA expression levels were assessed by reverse transcription‑quantitative polymerase chain reaction. (B) 14‑3‑3β protein expression levels were assessed by western blotting. (C) Quantification of protein expression relative to β‑actin (loading control). All experiments were repeated three times with three replicates each. All data are presented as mean ± standard deviation. *and #P<0.05 knockdown group vs. blank group and control group, respectively.

Article Snippet: Human osteoblastic cell line (hFOB1.19) and osteosarcoma cell lines (U2OS, Saos-2 and MG63) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Knockdown, Transfection, Control, Expressing, Polymerase Chain Reaction, Western Blot, Standard Deviation