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Bethyl rabbit polyclonal
Rabbit Polyclonal, supplied by Bethyl, used in various techniques. Bioz Stars score: 93/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+polyclonal+anti+fen1/Fen1+Antibody/pm39792662-241-47-102
Average 93 stars, based on 32 article reviews

rabbit polyclonal - by Bioz Stars, 2026-09

93/100 stars

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Article Title: FEN1 Ensures Telomere Stability by Facilitating Replication Fork Re-initiation
Article Snippet: Antibodies used: rabbit polyclonal anti-FEN1 (#586, Bethyl Laboratories, Montgomery, TX), mouse monoclonal anti-Actin (ABCAM, Cambridge, MA), rabbit polyclonal anti-TRF2 (H-300; Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-FLAG M2 (Sigma), rabbit polyclonal anti-cyclophilin A (Cell Signaling Technology, Danvers, MA).

Article Title: Mapping the FEN1 interaction domain with hTERT
Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-FEN1 (Bethyl), monoclonal anti-Myc (Santa Cruz), monoclonal anti-FLAG (Sigma).

Article Title: Flap Endonuclease 1 Limits Telomere Fragility on the Leading Strand
Article Snippet: The following antibodies were used: mouse monoclonal anti-Chk1 (sc8408, Santa Cruz Biotechnology, Santa Cruz, CA); rabbit monoclonal anti-Chk1, phospho-Ser-345 (2348, Cell Signaling Technology, Danvers, MA); rabbit polyclonal anti-FEN1 (A300–255A, Bethyl Laboratories, Montgomery, TX); mouse monoclonal anti-RNase H1 (H00246243-M01, Novus Biologicals, Littleton, CO); rat monoclonal anti-α-tubulin (ab6160, Abcam, Cambridge, UK); mouse monoclonal anti-β-catenin (610154, BD Biosciences); rabbit polyclonal anti-γH2AX (07-164, Millipore, Billerica, MA).

Article Title: Flap Endonuclease 1 Limits Telomere Fragility on the Leading Strand
Article Snippet: The following antibodies were used: mouse monoclonal anti-Chk1 (sc8408, Santa Cruz Biotechnology, Santa Cruz, CA); rabbit monoclonal anti-Chk1, phospho-Ser-345 (2348, Cell Signaling Technology, Danvers, MA); rabbit polyclonal anti-FEN1 (A300 –255A, Bethyl Laboratories, Montgomery, TX); mouse monoclonal anti-RNase H1 (H00246243-M01, Novus Biologicals, Littleton, CO); rat monoclonal anti- -tubulin (ab6160, Abcam, Cambridge, UK); mouse monoclonal anti- -catenin (610154, BD Biosciences); rabbit polyclonal anti- H2AX (07-164, Millipore, Billerica, MA).

Article Title: Mapping the FEN1 interaction domain with hTERT
Article Snippet: 2.2 Antibodies The following primary antibodies were used: rabbit polyclonal anti-FEN1 (Bethyl), monoclonal anti-Myc (Santa Cruz), monoclonal anti-FLAG (Sigma).

Image Search Results

hFen1 immunodepletion increased SP-BER product. ( A ) The BER patch size for a U:A and rAP:A plasmid substrate generated by asynchronous HEK 293 FT WCE or WCE supplemented with Polβ (50 nM) and aphidicolin (100 μg/ml). Aph, aphidicolin. **** indicates P < .0001. n = 3 independent experiments and the data are means ± SD. Async, asynchronous. ( B ) The hFen1 abundance in asynchronous HEK 293 FT WCE and anti-hFen1 antibody immunodepleted WCE were determined using western blot. ID: Immunodepletion. ( C ) The BER patch size for a U:A plasmid substrate generated by asynchronous HEK 293 FT WCE, hFen1 immunodepleted WCE, hFen1 (150 nM) supplemented hFen1 immunodepleted WCE, and xFen1 (150 nM) supplemented hFen1 immunodepleted WCE. **** indicates P < .0001. ns, not significant. n = 3 independent experiments and the data are means ± SD.

Journal: Nucleic Acids Research

Article Title: Pathways and products of base excision DNA repair in Xenopus laevis eggs: contrast with human cell pathways

doi: 10.1093/nar/gkaf1326

Figure Lengend Snippet: hFen1 immunodepletion increased SP-BER product. ( A ) The BER patch size for a U:A and rAP:A plasmid substrate generated by asynchronous HEK 293 FT WCE or WCE supplemented with Polβ (50 nM) and aphidicolin (100 μg/ml). Aph, aphidicolin. **** indicates P < .0001. n = 3 independent experiments and the data are means ± SD. Async, asynchronous. ( B ) The hFen1 abundance in asynchronous HEK 293 FT WCE and anti-hFen1 antibody immunodepleted WCE were determined using western blot. ID: Immunodepletion. ( C ) The BER patch size for a U:A plasmid substrate generated by asynchronous HEK 293 FT WCE, hFen1 immunodepleted WCE, hFen1 (150 nM) supplemented hFen1 immunodepleted WCE, and xFen1 (150 nM) supplemented hFen1 immunodepleted WCE. **** indicates P < .0001. ns, not significant. n = 3 independent experiments and the data are means ± SD.

Article Snippet: Rabbit polyclonal anti-hFen1 antibody (1:500, Proteintech, 14768-1-AP), rabbit polyclonal anti-xFen1 antibody (1:1000, see the “Acknowledgments” section), rabbit polyclonal anti-hPolβ antibody (1:250, Novus, NBP2-38600), rabbit polyclonal anti-GAPDH antibody (1:1000, Rockland, 600-401-A33), and IRDye 800CW labeled goat anti-rabbit IgG (1:10 000, LI-COR, 926-32211) were diluted in 1% non-fat milk in TBS-T.

Techniques: Immunodepletion, Plasmid Preparation, Generated, Western Blot

Selection and validation of FEN1 and ENDOU as candidate biomarkers. (A) Forest plot illustrating differential expression of FEN1 across studies integrated using MetaVolcanoR. (B) FEN1 transcript expression in TCGA-CESC cohort. (C) Forest plot illustrating differential expression of ENDOU across studies integrated using MetaVolcanoR. (D) Validation of ENDOU expression in TCGA-CESC cohort. For each box plot and Kruskal-Wallis test, the central line represents the median; the outer black lines on the box represent the upper and lower quartiles; dots represent outliers; the black lines are the whiskers, which extend from the interquartile ranges to the maximum values that are not classed as outliers. FEN1, flap structure-specific endonuclease 1;ENDOU, poly (U)-specific endoribonuclease; TCGA, The Cancer Genome Atlas; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; SCC, squamous cell carcinoma; ADC, adenocarcinoma; FC, fold change; CI, confidence interval.

Journal: Oncology Letters

Article Title: Integrative meta-analysis of gene expression profiles identifies FEN1 and ENDOU as potential diagnostic biomarkers for cervical squamous cell carcinoma

doi: 10.3892/ol.2021.13101

Figure Lengend Snippet: Selection and validation of FEN1 and ENDOU as candidate biomarkers. (A) Forest plot illustrating differential expression of FEN1 across studies integrated using MetaVolcanoR. (B) FEN1 transcript expression in TCGA-CESC cohort. (C) Forest plot illustrating differential expression of ENDOU across studies integrated using MetaVolcanoR. (D) Validation of ENDOU expression in TCGA-CESC cohort. For each box plot and Kruskal-Wallis test, the central line represents the median; the outer black lines on the box represent the upper and lower quartiles; dots represent outliers; the black lines are the whiskers, which extend from the interquartile ranges to the maximum values that are not classed as outliers. FEN1, flap structure-specific endonuclease 1;ENDOU, poly (U)-specific endoribonuclease; TCGA, The Cancer Genome Atlas; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; SCC, squamous cell carcinoma; ADC, adenocarcinoma; FC, fold change; CI, confidence interval.

Article Snippet: Antigen retrieval was performed with Tris-EDTA buffer pH 7.8 (Cell Conditioner #1; Ventana Medical System, Inc.) at 95°C for 44 min followed by blocking of endogenous peroxides and proteins with inhibitor CM (Ventana Medical System, Inc.; cat. no. 760-159) at 37°C for 4 min. IHC staining was performed as a fully automated assay in the BenchMark ULTRA automated slide stainer (Ventana Medical System, Inc.) using rabbit polyclonal antibody against FEN1 (1 ng/ml; Atlas antibodies cat. no. HPA006748; http://www.atlasantibodies.com ), ENDOU (2 ng/ml; Atlas antibodies, cat. no. HPA012388; http://www.atlasantibodies.com ), p16 (p16 CINtec ® Histology kit; Roche Diagnostics GmbH; cat no. 06695256001; http://diagnostics.roche.com ) and UltraView DAB IHC Detection kit (cat. no. 760-500; Ventana Medical System, Inc.).

Techniques: Selection, Biomarker Discovery, Quantitative Proteomics, Expressing

Expression of candidate biomarkers associated with HPV status and genotype. (A) Clustering mRNA expression of the candidate biomarkers based on HPV infection status. (B) Clustering mRNA expression of the candidate biomarkers based on HPV genotype. (C) Pearson's correlation coefficient analysis plot of p16 score and FEN1 expression in SCC tissues. (D) Pearson's correlation coefficient analysis plot of Ki67 score and FEN1 expression in SCC tissues. For each box plot and Kruskal-Wallis test, the central line represents the median; the outer black lines on the box represent the upper and lower quartiles; dots represent outliers; the black lines are the whiskers, which extend from the interquartile ranges to the maximum values that are not classed as outliers. P-values for each Kruskal-Wallis test are given on the plot. HPV, human papillomavirus; FEN1, flap structure-specific endonuclease 1; SCC, squamous cell carcinoma; ENDOU, poly (U)-specific endoribonuclease; Neg, negative; Pos, positive.

Journal: Oncology Letters

Article Title: Integrative meta-analysis of gene expression profiles identifies FEN1 and ENDOU as potential diagnostic biomarkers for cervical squamous cell carcinoma

doi: 10.3892/ol.2021.13101

Figure Lengend Snippet: Expression of candidate biomarkers associated with HPV status and genotype. (A) Clustering mRNA expression of the candidate biomarkers based on HPV infection status. (B) Clustering mRNA expression of the candidate biomarkers based on HPV genotype. (C) Pearson's correlation coefficient analysis plot of p16 score and FEN1 expression in SCC tissues. (D) Pearson's correlation coefficient analysis plot of Ki67 score and FEN1 expression in SCC tissues. For each box plot and Kruskal-Wallis test, the central line represents the median; the outer black lines on the box represent the upper and lower quartiles; dots represent outliers; the black lines are the whiskers, which extend from the interquartile ranges to the maximum values that are not classed as outliers. P-values for each Kruskal-Wallis test are given on the plot. HPV, human papillomavirus; FEN1, flap structure-specific endonuclease 1; SCC, squamous cell carcinoma; ENDOU, poly (U)-specific endoribonuclease; Neg, negative; Pos, positive.

Techniques: Expressing, Infection

Diagnostic and prognostic performance of FEN1 and ENDOU transcripts. (A) ROC curve analysis for FEN1 in the GSE13380 cervical cancer cohort. (B) ROC curve analysis for ENDOU in the GSE13380 cervical cancer cohort. (C) Overall survival analysis for FEN1 in the TCGA-CESC-SCC cohort. mRNA levels were dichotomized based on the median separation. (D) Overall survival analysis for ENDOU in TCGA-CESC-SCC cohort. mRNA levels were dichotomized based on the median separation. (E) Correlation between FEN1 expression and tumor infiltration signature in TCGA-CESC. (F) Correlation between ENDOU expression and tumor infiltration signature in TCGA-CESC. FEN1, flap structure-specific endonuclease 1; ENDOU, poly (U)-specific endoribonuclease; ROC, receiver operating characteristic; TCGA, The Cancer Genome Atlas; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; SCC, squamous cell carcinoma; AUC, area under the curve; CI, confidence interval; HR, hazard ratio.

Journal: Oncology Letters

Article Title: Integrative meta-analysis of gene expression profiles identifies FEN1 and ENDOU as potential diagnostic biomarkers for cervical squamous cell carcinoma

doi: 10.3892/ol.2021.13101

Figure Lengend Snippet: Diagnostic and prognostic performance of FEN1 and ENDOU transcripts. (A) ROC curve analysis for FEN1 in the GSE13380 cervical cancer cohort. (B) ROC curve analysis for ENDOU in the GSE13380 cervical cancer cohort. (C) Overall survival analysis for FEN1 in the TCGA-CESC-SCC cohort. mRNA levels were dichotomized based on the median separation. (D) Overall survival analysis for ENDOU in TCGA-CESC-SCC cohort. mRNA levels were dichotomized based on the median separation. (E) Correlation between FEN1 expression and tumor infiltration signature in TCGA-CESC. (F) Correlation between ENDOU expression and tumor infiltration signature in TCGA-CESC. FEN1, flap structure-specific endonuclease 1; ENDOU, poly (U)-specific endoribonuclease; ROC, receiver operating characteristic; TCGA, The Cancer Genome Atlas; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; SCC, squamous cell carcinoma; AUC, area under the curve; CI, confidence interval; HR, hazard ratio.

Techniques: Diagnostic Assay, Expressing

Clinical validation of FEN1 and ENDOU in cervical biopsies. (A) Frequency distribution of FEN1 IHC scores in normal and SCC tissues. (B) Frequency distribution of FEN1 IHC scores across SCC grades. (C) Frequency distribution of p16 IHC scores in normal and SCC tissues. (D) Frequency distribution of p16 IHC scores across SCC grades. (E) Frequency distribution of ENDOU immunohistochemistry scores in normal and SCC tissues. (F) IHC analysis of FEN1, ENDOUand p16 expression levels. FEN1, flap structure-specific endonuclease 1;ENDOU, poly (U)-specific endoribonuclease; IHC, immunohistochemistry; SCC, squamous cell carcinoma.

Journal: Oncology Letters

Article Title: Integrative meta-analysis of gene expression profiles identifies FEN1 and ENDOU as potential diagnostic biomarkers for cervical squamous cell carcinoma

doi: 10.3892/ol.2021.13101

Figure Lengend Snippet: Clinical validation of FEN1 and ENDOU in cervical biopsies. (A) Frequency distribution of FEN1 IHC scores in normal and SCC tissues. (B) Frequency distribution of FEN1 IHC scores across SCC grades. (C) Frequency distribution of p16 IHC scores in normal and SCC tissues. (D) Frequency distribution of p16 IHC scores across SCC grades. (E) Frequency distribution of ENDOU immunohistochemistry scores in normal and SCC tissues. (F) IHC analysis of FEN1, ENDOUand p16 expression levels. FEN1, flap structure-specific endonuclease 1;ENDOU, poly (U)-specific endoribonuclease; IHC, immunohistochemistry; SCC, squamous cell carcinoma.

Techniques: Biomarker Discovery, Immunohistochemistry, Expressing

Association between FEN1 expression and the clinicopathological characteristic.

Journal: Oncology Letters

Article Title: Integrative meta-analysis of gene expression profiles identifies FEN1 and ENDOU as potential diagnostic biomarkers for cervical squamous cell carcinoma

doi: 10.3892/ol.2021.13101

Figure Lengend Snippet: Association between FEN1 expression and the clinicopathological characteristic.

Techniques: Expressing

Diagnostic accuracy of FEN1 staining and comparator tests.

Journal: Oncology Letters

Article Title: Integrative meta-analysis of gene expression profiles identifies FEN1 and ENDOU as potential diagnostic biomarkers for cervical squamous cell carcinoma

doi: 10.3892/ol.2021.13101

Figure Lengend Snippet: Diagnostic accuracy of FEN1 staining and comparator tests.

Techniques: Diagnostic Assay, Staining

Potential function of FEN1 and ENDOU in cervical carcinogenesis. (A) Pearson's correlation coefficient analysis plot of G 2 /M score and FEN1 expression in SCC tissues. (B) FEN1 concept network for prediction of gene function. (C) Ridge plot illustrating GO enrichment in cervical tumors expressing high levels of FEN1. (D) Correlation matrix between FEN1 and phase-specific cyclins, CDKs and regulators. (E) Correlation matrix between ENDOU and different cytokeratins. (F) Ridge plot illustrating Gene Set Enrichment Analysis, GO enrichment in cervical tumors expressing high levels of ENDOU. FEN1, flap structure-specific endonuclease 1; ENDOU, poly (U)-specific endoribonuclease; SCC, squamous cell carcinoma; GO, Gene Ontology.

Journal: Oncology Letters

Article Title: Integrative meta-analysis of gene expression profiles identifies FEN1 and ENDOU as potential diagnostic biomarkers for cervical squamous cell carcinoma

doi: 10.3892/ol.2021.13101

Figure Lengend Snippet: Potential function of FEN1 and ENDOU in cervical carcinogenesis. (A) Pearson's correlation coefficient analysis plot of G 2 /M score and FEN1 expression in SCC tissues. (B) FEN1 concept network for prediction of gene function. (C) Ridge plot illustrating GO enrichment in cervical tumors expressing high levels of FEN1. (D) Correlation matrix between FEN1 and phase-specific cyclins, CDKs and regulators. (E) Correlation matrix between ENDOU and different cytokeratins. (F) Ridge plot illustrating Gene Set Enrichment Analysis, GO enrichment in cervical tumors expressing high levels of ENDOU. FEN1, flap structure-specific endonuclease 1; ENDOU, poly (U)-specific endoribonuclease; SCC, squamous cell carcinoma; GO, Gene Ontology.

Techniques: Expressing

$(A) Left , individual data points and mean (±SD) doubling time for wild type (WT) and FEN1 −/− DT40 cells from 9 independent experiments. Statistical significance was assessed by paired t test. Middle , representative cell cycle distribution profiles. Right , quantification of the fraction of cells with sub-G1 content. Data represent the mean (±SD) from 3 independent experiments with individual data points plotted. (B) Representative ScanR image galleries of data quantified in , of anti-poly(ADP-ribose) immunofluorescence (PAR) (MABE1031) in wild type (WT) and FEN1 −/− DT40 cells (only PARGi-treated cells are shown).$

Journal: bioRxiv

Article Title: PARP Inhibition Impedes the Maturation of Nascent DNA Strands During DNA Replication

doi: 10.1101/2021.07.03.450982

Figure Lengend Snippet: (A) Left , individual data points and mean (±SD) doubling time for wild type (WT) and FEN1 −/− DT40 cells from 9 independent experiments. Statistical significance was assessed by paired t test. Middle , representative cell cycle distribution profiles. Right , quantification of the fraction of cells with sub-G1 content. Data represent the mean (±SD) from 3 independent experiments with individual data points plotted. (B) Representative ScanR image galleries of data quantified in , of anti-poly(ADP-ribose) immunofluorescence (PAR) (MABE1031) in wild type (WT) and FEN1 −/− DT40 cells (only PARGi-treated cells are shown).

Article Snippet: Primary antibodies used are as follows: Anti-poly-ADP-ribose binding reagent/ PAR reagent (recombinant protein fused to rabbit Fc tag; Millipore, MABE1031), Rabbit monoclonal anti-Poly/Mono-ADP Ribose (anti-PAR/MAR, Cell Signaling, 83732), Mouse monoclonal anti-PCNA (Santa Cruz, sc-56), Rabbit monoclonal anti-PARP1 (Cell Signaling, 9532), Rat polyclonal anti-α-tubulin (Abcam, ab6160), Rabbit polyclonal anti-H3 (Abcam, ab1791), Rabbit polyclonal anti-FEN1 (LifeSpan Biosciences, LS-C80825), Mouse monoclonal anti-biotin (Merck, BN-34), Mouse monoclonal anti-PARP1 (Santa Cruz, sc-8007), Rat recombinant anti-PCNA (Abcam, ab252848), Rat monoclonal anti-BrdU (Abcam, ab6326), Mouse monoclonal anti-BrdU (Becton Dickinson, 347580), Mouse monoclonal anti-ssDNA (Millipore, MAB3034).

Techniques: Immunofluorescence

$(A) Representative images ( left ) and ScanR quantification ( right ) of poly(ADP-ribose) fluorescence (PAR) (MABE1031) in wild type (WT) and FEN1 −/− DT40 cells incubated for 30 min with DMSO vehicle (−) or with 10 μM PARG inhibitor (PARGi) to prevent poly(ADP-ribose) degradation. S phase cells were distinguished by PCNA staining and G1/G2 phase cells by DAPI intensity. Data represent the average (±SD) mean PAR fluorescence in arbitrary units (AU) from 3 independent experiments with individual data points plotted. Scale bars, 20 μm. Statistical significance was assessed by 1-way ANOVA with post-hoc Sidak’s multiple comparisons test. Galleries of representative cells from ScanR analysis are shown in Figure S1B. (B) Western blots ( left ) of PARP1, PCNA, tubulin, and histone H3 (H3) in soluble (detergent extracted) and chromatin-containing fractions from wild type (WT) and FEN1 −/− DT40 cells. Cells were incubated for 30 min with DMSO vehicle (NT) or 10 μM PARP inhibitor (PARPi; Olaparib) prior to cell fractionation. For quantification of PARP1 levels in chromatin ( right ), PARP1 protein levels in the detergent-insoluble material were first normalised to ponceau S-stained histone levels and then expressed relative to the PARP1 level in untreated wild type chromatin. Data represent the mean (±SD) of 6 independent experiments with individual data points plotted. Statistical significance was assessed by Student’s paired t test. (C) Clonogenic survival assays for wild type (WT), FEN1 −/− , and XRCC3 −/− DT40 cells grown with the indicated concentrations of PARPi (Olaparib). Data represent the mean (±SD) of 4 independent experiments. Statistical significance was assessed by 2-way ANOVA with post-hoc Tukey’s multiple comparisons test. For all panels; ns, not significant; *p< 0.05; **p<0.01; ****p<0.0001.$

Journal: bioRxiv

Article Title: PARP Inhibition Impedes the Maturation of Nascent DNA Strands During DNA Replication

doi: 10.1101/2021.07.03.450982

Figure Lengend Snippet: (A) Representative images ( left ) and ScanR quantification ( right ) of poly(ADP-ribose) fluorescence (PAR) (MABE1031) in wild type (WT) and FEN1 −/− DT40 cells incubated for 30 min with DMSO vehicle (−) or with 10 μM PARG inhibitor (PARGi) to prevent poly(ADP-ribose) degradation. S phase cells were distinguished by PCNA staining and G1/G2 phase cells by DAPI intensity. Data represent the average (±SD) mean PAR fluorescence in arbitrary units (AU) from 3 independent experiments with individual data points plotted. Scale bars, 20 μm. Statistical significance was assessed by 1-way ANOVA with post-hoc Sidak’s multiple comparisons test. Galleries of representative cells from ScanR analysis are shown in Figure S1B. (B) Western blots ( left ) of PARP1, PCNA, tubulin, and histone H3 (H3) in soluble (detergent extracted) and chromatin-containing fractions from wild type (WT) and FEN1 −/− DT40 cells. Cells were incubated for 30 min with DMSO vehicle (NT) or 10 μM PARP inhibitor (PARPi; Olaparib) prior to cell fractionation. For quantification of PARP1 levels in chromatin ( right ), PARP1 protein levels in the detergent-insoluble material were first normalised to ponceau S-stained histone levels and then expressed relative to the PARP1 level in untreated wild type chromatin. Data represent the mean (±SD) of 6 independent experiments with individual data points plotted. Statistical significance was assessed by Student’s paired t test. (C) Clonogenic survival assays for wild type (WT), FEN1 −/− , and XRCC3 −/− DT40 cells grown with the indicated concentrations of PARPi (Olaparib). Data represent the mean (±SD) of 4 independent experiments. Statistical significance was assessed by 2-way ANOVA with post-hoc Tukey’s multiple comparisons test. For all panels; ns, not significant; *p< 0.05; **p<0.01; ****p<0.0001.

Techniques: Fluorescence, Incubation, Staining, Western Blot, Cell Fractionation

(A) DNA strand breaks were quantified by alkaline comet assays in wild type (WT) and FEN1 −/− DT40 cells following a 2 h incubation with DMSO vehicle (−) or 10 μM PARPi (KU 0058948). Genomic DNA was scored for comet tail moments by staining with SYBR Green. For each sample, scatter plots are the comet tail moments (an arbitrary unit of DNA strand breakage) of 300 cells combined from 3 independent experiments (100 cells per sample per experiment) and the bars represent the median and interquartile range. The individual data sets from the three experimental repeats are plotted in Figure S2A. Statistical significance was determined by 2-way ANOVA with Tukey’s post hoc multiple comparisons test, with individual tail moments within an experiment treated as technical replicates and with experimental matching/blocking by experimental repeat. (B) DNA strand breaks in total genomic DNA were quantified by alkaline comet assays in S phase and non-S phase cells following incubation or not with 10 μM PARPi (Olaparib) for 2 h. S phase cells were identified by labelling with BrdU for the final 45 min. Genomic DNA was scored for comet tail moments by staining with propidium iodide (PI). For each sample, scatter plots are the comet tail moments of 100 cells combined from 2 independent experiments (50 cells per sample per experiment) and the bars represent the median and interquartile range. The individual data sets from the two experimental repeats are plotted in Figure S2B, and statistical analysis was conducted as in panel A. (C) DNA breaks in nascent DNA strands quantified in DT40 cells by anti-BrdU alkaline comet assays after pulse labelling (30 min) with BrdU followed by a subsequent chase (90 min), as indicated in the schematic ( left ). Where indicated, 10 μM PARPi (Olaparib) was present in the media. Alkaline comet tail moments were quantified in BrdU-labelled nascent single-strands by staining with anti-BrdU antibodies (“BrdU comet tail moment”). For each sample, scatter plots show BrdU comet tail moments from 300 cells combined from 3 independent experiments (100 cells per sample per experiment) and bars are the median and interquartile range. The individual data sets from the three experimental repeats are plotted separately in Figure S2D, and statistical analysis was conducted as in panel A. For all panels; ns, not significant; *p< 0.05; ****p<0.0001.

Journal: bioRxiv

Article Title: PARP Inhibition Impedes the Maturation of Nascent DNA Strands During DNA Replication

doi: 10.1101/2021.07.03.450982

Figure Lengend Snippet: (A) DNA strand breaks were quantified by alkaline comet assays in wild type (WT) and FEN1 −/− DT40 cells following a 2 h incubation with DMSO vehicle (−) or 10 μM PARPi (KU 0058948). Genomic DNA was scored for comet tail moments by staining with SYBR Green. For each sample, scatter plots are the comet tail moments (an arbitrary unit of DNA strand breakage) of 300 cells combined from 3 independent experiments (100 cells per sample per experiment) and the bars represent the median and interquartile range. The individual data sets from the three experimental repeats are plotted in Figure S2A. Statistical significance was determined by 2-way ANOVA with Tukey’s post hoc multiple comparisons test, with individual tail moments within an experiment treated as technical replicates and with experimental matching/blocking by experimental repeat. (B) DNA strand breaks in total genomic DNA were quantified by alkaline comet assays in S phase and non-S phase cells following incubation or not with 10 μM PARPi (Olaparib) for 2 h. S phase cells were identified by labelling with BrdU for the final 45 min. Genomic DNA was scored for comet tail moments by staining with propidium iodide (PI). For each sample, scatter plots are the comet tail moments of 100 cells combined from 2 independent experiments (50 cells per sample per experiment) and the bars represent the median and interquartile range. The individual data sets from the two experimental repeats are plotted in Figure S2B, and statistical analysis was conducted as in panel A. (C) DNA breaks in nascent DNA strands quantified in DT40 cells by anti-BrdU alkaline comet assays after pulse labelling (30 min) with BrdU followed by a subsequent chase (90 min), as indicated in the schematic ( left ). Where indicated, 10 μM PARPi (Olaparib) was present in the media. Alkaline comet tail moments were quantified in BrdU-labelled nascent single-strands by staining with anti-BrdU antibodies (“BrdU comet tail moment”). For each sample, scatter plots show BrdU comet tail moments from 300 cells combined from 3 independent experiments (100 cells per sample per experiment) and bars are the median and interquartile range. The individual data sets from the three experimental repeats are plotted separately in Figure S2D, and statistical analysis was conducted as in panel A. For all panels; ns, not significant; *p< 0.05; ****p<0.0001.

Techniques: Incubation, Staining, SYBR Green Assay, Blocking Assay

(A, B) Scatter plots of the data from & , respectively, with each experimental repeat plotted side by side. (C) DNA strand breaks quantified by alkaline comet assays in wild type (WT) and FEN1 −/− DT40 cells pulse-labelled or not with BrdU for the last 45 min of a 2h-incubation with DMSO vehicle or 10 μM PARPi (Olaparib). Genomic DNA was visualised for scoring comet tail moments by staining with propidium iodide (PI). Plotted data are the individual comet tail moments from 2 independent experiments (100 cells per sample per experiment) and the bars are the median and interquartile range. (D) Scatter plots of the data from with each experimental repeat plotted side by side. (E) γ-ray calibration curve for BrdU alkaline comet assays in DT40. Wild type DT40 cells were incubated with media containing BrdU (100 μM) for 20 h to fully label genomic DNA and then treated with the indicated dose of γ-rays. Alkaline comet tail moments of anti-BrdU stained DNA were quantified under the same electrophoretic and experimental conditions as those applied in BrdU pulse-labelled cells in . The average (±SD) of the median comet tail moments from 2 independent experiments (50 cells per sample per experiment) are plotted. The average fragment size of single-stranded DNA following the indicated γ-ray dose is indicated, calculated on the assumption that each Gy induces ~1100 total DNA breaks (~1000 SSBs & ~50 DSBs) – per diploid human genome (12×10 9 nucleotides) and thus ~1 break every 1×10 7 nucleotides. Notice that the BrdU alkaline tail moments are insensitive to average fragment sizes below 500 kb, perhaps because fragments below this size are ‘lost’ from the assay during alkaline lysis and/or electrophoresis.

Journal: bioRxiv

Article Title: PARP Inhibition Impedes the Maturation of Nascent DNA Strands During DNA Replication

doi: 10.1101/2021.07.03.450982

Figure Lengend Snippet: (A, B) Scatter plots of the data from & , respectively, with each experimental repeat plotted side by side. (C) DNA strand breaks quantified by alkaline comet assays in wild type (WT) and FEN1 −/− DT40 cells pulse-labelled or not with BrdU for the last 45 min of a 2h-incubation with DMSO vehicle or 10 μM PARPi (Olaparib). Genomic DNA was visualised for scoring comet tail moments by staining with propidium iodide (PI). Plotted data are the individual comet tail moments from 2 independent experiments (100 cells per sample per experiment) and the bars are the median and interquartile range. (D) Scatter plots of the data from with each experimental repeat plotted side by side. (E) γ-ray calibration curve for BrdU alkaline comet assays in DT40. Wild type DT40 cells were incubated with media containing BrdU (100 μM) for 20 h to fully label genomic DNA and then treated with the indicated dose of γ-rays. Alkaline comet tail moments of anti-BrdU stained DNA were quantified under the same electrophoretic and experimental conditions as those applied in BrdU pulse-labelled cells in . The average (±SD) of the median comet tail moments from 2 independent experiments (50 cells per sample per experiment) are plotted. The average fragment size of single-stranded DNA following the indicated γ-ray dose is indicated, calculated on the assumption that each Gy induces ~1100 total DNA breaks (~1000 SSBs & ~50 DSBs) – per diploid human genome (12×10 9 nucleotides) and thus ~1 break every 1×10 7 nucleotides. Notice that the BrdU alkaline tail moments are insensitive to average fragment sizes below 500 kb, perhaps because fragments below this size are ‘lost’ from the assay during alkaline lysis and/or electrophoresis.

Techniques: Incubation, Staining, Alkaline Lysis, Electrophoresis

$(A) Schematic for measuring the size distribution of 3 H-labelled nascent DNA strands in wild type (WT) and FEN1 −/− DT40 cells following 10 min pulse labelling with 3 H-thymidine and during a subsequent 20 min cold chase, in the absence or presence of 10 μM PARPi (Olaparib) as indicated, by alkaline agarose gel electrophoresis, (B) Quantification of 3 H-pulse-labelled nascent DNA strands in the size ranges <0.5 kb, 0.5-10 kb, and >10 kb by liquid scintillation counting of radioactivity in alkaline agarose gel slices. Graphs show the mean fraction (%) ±SD of 3 H radioactivity (in CPM) in nascent DNA fragments of the indicated sizes from 3 independent experiments. Statistical analysis of the fraction of radioactivity detected as nascent DNA strands of <10 kb was conducted by 2-way ANOVA with Tukey’s post hoc multiple comparisons test.$

Journal: bioRxiv

Article Title: PARP Inhibition Impedes the Maturation of Nascent DNA Strands During DNA Replication

doi: 10.1101/2021.07.03.450982

Figure Lengend Snippet: (A) Schematic for measuring the size distribution of 3 H-labelled nascent DNA strands in wild type (WT) and FEN1 −/− DT40 cells following 10 min pulse labelling with 3 H-thymidine and during a subsequent 20 min cold chase, in the absence or presence of 10 μM PARPi (Olaparib) as indicated, by alkaline agarose gel electrophoresis, (B) Quantification of 3 H-pulse-labelled nascent DNA strands in the size ranges <0.5 kb, 0.5-10 kb, and >10 kb by liquid scintillation counting of radioactivity in alkaline agarose gel slices. Graphs show the mean fraction (%) ±SD of 3 H radioactivity (in CPM) in nascent DNA fragments of the indicated sizes from 3 independent experiments. Statistical analysis of the fraction of radioactivity detected as nascent DNA strands of <10 kb was conducted by 2-way ANOVA with Tukey’s post hoc multiple comparisons test.

Techniques: Agarose Gel Electrophoresis, Radioactivity

(A) Schematic for measuring the rates of DNA replication fork progression during consecutive CldU and IdU pulse labelling by DNA combing in wild type (WT) and FEN1 −/− DT40 cells incubated or not as indicated in 10 μM PARPi (Olaparib). (B) CldU tract-lengths in dual-labelled DNA fibres were measured in 3 independent experiments (>65 fibres per sample per experiment) and the distributions of CldU fork speeds (calculated assuming a constant stretching factor of 2kb/μm) are presented as scatter plots. Bars depict the median and interquartile range. Individual data points from the three experimental repeats are plotted separately in Figure S3A and statistical analysis was conducted by 2-way ANOVA (mixed effects model) with Tukey’s post hoc multiple comparisons test. (C) IdU fork speeds in dual-labelled DNA fibres scored and analysed as in panel B. Individual data points from the three experimental repeats are plotted separately in Figure S3B. (D) The ratio of IdU and CldU tract lengths in dual-labelled fibres, presented as distributions in scatter plots and analysed as in panel B. Individual data points from the three experimental repeats are plotted separately in Figure S3C. For all panels; ns, not significant; ****p<0.0001.

Journal: bioRxiv

Article Title: PARP Inhibition Impedes the Maturation of Nascent DNA Strands During DNA Replication

doi: 10.1101/2021.07.03.450982

Figure Lengend Snippet: (A) Schematic for measuring the rates of DNA replication fork progression during consecutive CldU and IdU pulse labelling by DNA combing in wild type (WT) and FEN1 −/− DT40 cells incubated or not as indicated in 10 μM PARPi (Olaparib). (B) CldU tract-lengths in dual-labelled DNA fibres were measured in 3 independent experiments (>65 fibres per sample per experiment) and the distributions of CldU fork speeds (calculated assuming a constant stretching factor of 2kb/μm) are presented as scatter plots. Bars depict the median and interquartile range. Individual data points from the three experimental repeats are plotted separately in Figure S3A and statistical analysis was conducted by 2-way ANOVA (mixed effects model) with Tukey’s post hoc multiple comparisons test. (C) IdU fork speeds in dual-labelled DNA fibres scored and analysed as in panel B. Individual data points from the three experimental repeats are plotted separately in Figure S3B. (D) The ratio of IdU and CldU tract lengths in dual-labelled fibres, presented as distributions in scatter plots and analysed as in panel B. Individual data points from the three experimental repeats are plotted separately in Figure S3C. For all panels; ns, not significant; ****p<0.0001.

Techniques: Incubation

(A) Analysis of wild type U2OS cells and the indicated FEN1 −/− clones by western blotting of whole cell extracts ( left ) and by Sanger sequencing ( right ) of PCR products of genomic DNA spanning the predicted Cas9 break site. The PAM is shown in green, gRNA targeted sequence is shown in yellow and mutated nucleotides are shown in pink.

Journal: bioRxiv

Article Title: PARP Inhibition Impedes the Maturation of Nascent DNA Strands During DNA Replication

doi: 10.1101/2021.07.03.450982

Figure Lengend Snippet: (A) Analysis of wild type U2OS cells and the indicated FEN1 −/− clones by western blotting of whole cell extracts ( left ) and by Sanger sequencing ( right ) of PCR products of genomic DNA spanning the predicted Cas9 break site. The PAM is shown in green, gRNA targeted sequence is shown in yellow and mutated nucleotides are shown in pink.

Techniques: Clone Assay, Western Blot, Sequencing

(A) PARP activity in wild type (WT) and FEN1 −/− U2OS cells (clones #15, #6, #16), incubated with or without PARG inhibitor (10 μM) for 30 min, measured by anti-ADP-ribose (MAR/PAR) (CST 83732) immunofluorescence in detergent extracted cells. Representative microscopy images (WT and FEN1 −/− clone #15 only) are shown left , and ScanR quantification (WT and all FEN1 −/− clones) is shown right . S phase cells were distinguished by positive PCNA staining and G1/G2 cells (PCNA-negative) by DNA content (measured with DAPI). Data represent the mean (±SD) total intensity of MAR/PAR (in arbitrary units (AU)) from 5 independent experiments with individual data points plotted. Statistical significance was assessed by 1-way ANOVA with post-hoc Sidak’s multiple comparisons test. (B) Physical proximity of newly incorporated EdU and ADP-ribose (MAR/PAR; CST 83732) in wild type (WT) and FEN1 −/− U2OS cells, measured following pulse labelling for 10 min and during a subsequent 30 min thymidine (THM) chase by PLA, as indicated in the schematic. Sampled cells were pre-extracted and fixed using PFA. PLA was measured using anti-biotin antibodies to detect biotin-azide clicked to EdU and anti-ADP-ribose antibodies to detect sites of PARP activity. Representative ScanR image galleries ( left , each box is a single cell) and quantification ( right ) are shown. Total PLA intensity in PCNA positive cells was normalized to that in wild type U2OS cells immediately after EdU pulse. Data represent mean (±SD) from 3-4 independent experiments with individual data points plotted. Statistical significance was assessed by 1-way ANOVA with post-hoc Sidak’s multiple comparisons test. (C) Quantification of ADP-ribose and EdU levels following pulse labelling and during the thymidine chase. Cells were treated and fixed as in panel A and EdU and ADP-ribose intensities were determined by ScanR in PCNA positive cells. Data represent mean (±SD) from 3-4 independent experiments with individual data points plotted. Statistical significance was assessed by 1-way ANOVA with post-hoc Sidak’s multiple comparisons test. For all panels; ns, not significant, *p< 0.05.

Journal: bioRxiv

Article Title: PARP Inhibition Impedes the Maturation of Nascent DNA Strands During DNA Replication

doi: 10.1101/2021.07.03.450982

Figure Lengend Snippet: (A) PARP activity in wild type (WT) and FEN1 −/− U2OS cells (clones #15, #6, #16), incubated with or without PARG inhibitor (10 μM) for 30 min, measured by anti-ADP-ribose (MAR/PAR) (CST 83732) immunofluorescence in detergent extracted cells. Representative microscopy images (WT and FEN1 −/− clone #15 only) are shown left , and ScanR quantification (WT and all FEN1 −/− clones) is shown right . S phase cells were distinguished by positive PCNA staining and G1/G2 cells (PCNA-negative) by DNA content (measured with DAPI). Data represent the mean (±SD) total intensity of MAR/PAR (in arbitrary units (AU)) from 5 independent experiments with individual data points plotted. Statistical significance was assessed by 1-way ANOVA with post-hoc Sidak’s multiple comparisons test. (B) Physical proximity of newly incorporated EdU and ADP-ribose (MAR/PAR; CST 83732) in wild type (WT) and FEN1 −/− U2OS cells, measured following pulse labelling for 10 min and during a subsequent 30 min thymidine (THM) chase by PLA, as indicated in the schematic. Sampled cells were pre-extracted and fixed using PFA. PLA was measured using anti-biotin antibodies to detect biotin-azide clicked to EdU and anti-ADP-ribose antibodies to detect sites of PARP activity. Representative ScanR image galleries ( left , each box is a single cell) and quantification ( right ) are shown. Total PLA intensity in PCNA positive cells was normalized to that in wild type U2OS cells immediately after EdU pulse. Data represent mean (±SD) from 3-4 independent experiments with individual data points plotted. Statistical significance was assessed by 1-way ANOVA with post-hoc Sidak’s multiple comparisons test. (C) Quantification of ADP-ribose and EdU levels following pulse labelling and during the thymidine chase. Cells were treated and fixed as in panel A and EdU and ADP-ribose intensities were determined by ScanR in PCNA positive cells. Data represent mean (±SD) from 3-4 independent experiments with individual data points plotted. Statistical significance was assessed by 1-way ANOVA with post-hoc Sidak’s multiple comparisons test. For all panels; ns, not significant, *p< 0.05.

Techniques: Activity Assay, Clone Assay, Incubation, Immunofluorescence, Microscopy, Staining

(A) DNA breaks in nascent DNA strands quantified in wild type (WT) and FEN1 −/− (clones #15 & #16) U2OS cells by alkaline comet assays after pulse labelling (30 min) with BrdU followed by a subsequent chase (90 min) as indicated in the schematic ( top ). 10 μM PARPi (Olaparib) was employed or not as indicated. Nascent DNA strands were scored for comet tail moments by staining with anti-BrdU antibodies. For each sample, scatter plots are anti-BrdU comet tail moments from 300 cells combined from 3 independent experiments (100 cells per sample per experiment) and bars are the median and interquartile range. The individual data sets from the three experimental repeats are plotted separately in Figure S5A, and statistical analysis was conducted as in . (B) DNA breaks in nascent DNA strands quantified in wild type RPE-1 cells by alkaline comet assays after pulse labelling (30 min) with BrdU followed by a subsequent chase (90 min) as indicated in the schematic ( top ). Where indicated, cells were incubated with 10 μM PARPi (Olaparib) and/or 10 μM FEN1 inhibitor (FEN1i). Nascent DNA strands were scored for comet tail moments by staining with anti-BrdU antibodies. Scatter plots are as in panel A, with separate experimental repeats plotted in Figure S5B. For all panels; ns, not significant; **p<0.01, ****p<0.0001.

Journal: bioRxiv

Article Title: PARP Inhibition Impedes the Maturation of Nascent DNA Strands During DNA Replication

doi: 10.1101/2021.07.03.450982

Figure Lengend Snippet: (A) DNA breaks in nascent DNA strands quantified in wild type (WT) and FEN1 −/− (clones #15 & #16) U2OS cells by alkaline comet assays after pulse labelling (30 min) with BrdU followed by a subsequent chase (90 min) as indicated in the schematic ( top ). 10 μM PARPi (Olaparib) was employed or not as indicated. Nascent DNA strands were scored for comet tail moments by staining with anti-BrdU antibodies. For each sample, scatter plots are anti-BrdU comet tail moments from 300 cells combined from 3 independent experiments (100 cells per sample per experiment) and bars are the median and interquartile range. The individual data sets from the three experimental repeats are plotted separately in Figure S5A, and statistical analysis was conducted as in . (B) DNA breaks in nascent DNA strands quantified in wild type RPE-1 cells by alkaline comet assays after pulse labelling (30 min) with BrdU followed by a subsequent chase (90 min) as indicated in the schematic ( top ). Where indicated, cells were incubated with 10 μM PARPi (Olaparib) and/or 10 μM FEN1 inhibitor (FEN1i). Nascent DNA strands were scored for comet tail moments by staining with anti-BrdU antibodies. Scatter plots are as in panel A, with separate experimental repeats plotted in Figure S5B. For all panels; ns, not significant; **p<0.01, ****p<0.0001.

Techniques: Clone Assay, Staining, Incubation

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