rabbit anti top3a  (Cell Signaling Technology Inc)

Journal: Cell reports

Article Title: PARP1-driven repair of topoisomerase IIIα DNA-protein crosslinks by FEN1

doi: 10.1016/j.celrep.2024.114522

Figure Lengend Snippet: (A) Top: outline of the experimental protocol for transfection followed by RADAR assay in MCF-7 cells with and without FEN1 inactivation and ectopic expression of TOP3A-R364W. Bottom (left): representative slot blot for TOP3Acc detection by RADAR assay from cells with the indicated genotypes. TOP3Accs were detected with anti-TOP3A antibody. Bottom (right): bar graph from quantitation of the 3 independent experiments as shown on the left. Error bars represent mean ± SD. p values were obtained from two-tailed unpaired t test with Welch’s correction. * p < 0.05. (B) Left panel: representative slot blot detecting endogenous TOP3Accs in shControl and sh FEN1 MCF-7 cells. TOP3Accs were detected by RADAR assay with anti-TOP3A antibody. Right: quantitation of TOP3Accs from three independent RADAR assays as shown on the left. Error bars represent mean ± SD. p values were obtained from two-tailed unpaired t test with Welch’s correction. ** p < 0.01. (C) Top: outline of the experimental protocol in HCT116 cells expressing TOP3A-R364W. Cells were transfected with either siControl or siFEN1 for 72 and 24 h with TOP3A-R364W for 48 h before performing RADAR assay to detect TOP3A-DPCs. Bottom (left): representative slot blot for TOP3Acc detection by RADAR assay from siControl and siFEN1 cells. TOP3Accs were detected with an anti-TOP3A antibody. Bottom (right): bar graph from quantitation of the 3 independent experiments as shown on the left. Error bars represent mean ± SD. p values were obtained from two-tailed unpaired t test with Welch’s correction. ** p < 0.01. (D) Top: HCT116 cells were transfected with the TOP3A-R364W expression plasmid for 48 h, followed by treatment with either FEN1i or DMSO (drug solvent) (10 μM, 1 h) before cell harvest. Cells were then subjected to the RADAR assay to detect TOP3A-DPCs by slot blot. DMSO was used in the control samples. Bottom (left): representative slot blot of RADAR assay from control and FEN1-inhibited cells. Bottom (right): bar graph from quantitation of the 3 independent experiments as shown on the left. Error bars represent mean ± SD. p values were obtained from two-tailed unpaired t test with Welch’s correction. ** p < 0.01. (E) Ectopic expression of FEN1 reduces TOP3Accs. Top left: outline of the experimental protocol. Bottom left: representative slot blot for MCF-7 cells first transfected with TOP3A-R364W and, 1 day later, transfected with FEN1-WT or the nuclease-dead mutant FEN1 (FEN1-D118A) plasmids. Cells were transfected in 6-well plates with 1.2 μg of each of the TOP3A-R364W and FEN1 plasmids (1:1 ratio). Representative RADAR assays with an anti-TOP3A antibody were used for TOP3A-DPC detection. EV, empty vector transfected. Right: bar graph from quantitation of 3 independent experiments as shown on the left. Error bars represent mean ± SD. p values were obtained from two-tailed unpaired t test with Welch’s correction. *** p < 0.001 (EV vs FEN1-WT) and ns: not significant (EV vs FEN1-D181A). In (A)–(E), the indicated amounts of DNA were loaded, and loading was further tested by slot blot and probing with an anti-double-stranded DNA (dsDNA) antibody.

Article Snippet: Nucleic acid containing protein adducts were quantitated, slot-blotted and TOP3A-DPCs were detected with rabbit polyclonal anti-TOP3A antibody (dilution 1:1000, Proteintech, Rosemont, IL, CAT#: 14525-1-AP).

Techniques: Transfection, Expressing, Dot Blot, Quantitation Assay, Two Tailed Test, Plasmid Preparation, Solvent, Control, Mutagenesis

Journal: Cell reports

Article Title: PARP1-driven repair of topoisomerase IIIα DNA-protein crosslinks by FEN1

doi: 10.1016/j.celrep.2024.114522

Figure Lengend Snippet: (A) PARP inhibition reduces FEN1 interaction with TOP3A. Top: protocol for the PLA experiments. Samples were prepared from U2OS cells non-transfected (NT) or transfected with FLAG-tagged TOP3A-WT or TOP3A-R364W for 48 h. Cells expressing FLAG-tagged TOP3A-R364W after 48 h transfection were treated with 1 μM talazoparib (PARPi) for 1 h before cell fixation and PLA. Bottom: representative images of a PLA for FLAG-tagged TOP3A and endogenous FEN1 for indicated conditions. Scale bar, 5 μm. Right: quantitation of the TOP3A-FLAG and FEN1 PLA foci shown in (A). Results are represented as SuperPlots showing individual data points in different colors, the average from each experiment, and the mean of the averages ± SD. **** p < 0.0001, ordinary one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test. (B) Representative FLAG immunoprecipitation (IP) assay showing the interaction of TOP3A with FEN1 and PARP1. Following transfection of FLAG-tagged empty vector (EV) or TOP3A-WT and TOP3A-R364W, U2OS cells were treated with either DMSO (drug solvent control), PARGi or PARPi alone (1 μM, 1 h), or a combined treatment of PARGi and PARPi (1 μM, 1 h each). Cells were lysed and chromatin fractions were immunoprecipitated with A-protein magnetic beads. FLAG IP under denaturing conditions was followed by immunoblotting with the indicated antibodies. (C)Representative images of a PLA for FLAG-tagged TOP3A and endogenous PAR in U2OS cells. PLA with FLAG + PAR antibody either in pretreatment of DMSO (drug solvent), PARGi or PARPi alone (1 μM, 1 h), or combined treatment of PARGi and PARPi (1 μM, 1 h each). DMSO was used in the control samples. Scale bar, 5 μm. (D) Quantitation of the TOP3A-FLAG and PAR PLA foci shown in (C). Results are represented as SuperPlots showing individual data points in different colors, the average from each experiment, and the mean of the averages ± SD. **** p < 0.0001, ordinary one-way ANOVA with Tukey’s multiple comparisons test. (E) Representative images of a PLA for FLAG-tagged TOP3A and endogenous PARP1 in U2OS cells expressing EV, TOP3A-WT, or TOP3A-R364W. PLA was performed with FLAG + PARP1 antibody in the presence of PARGi (1 μM, 1 h). Scale bar, 5 μm. (F) Quantitation of the TOP3A-FLAG and PARP1 PLA foci shown in (E). Results are represented as SuperPlots showing individual data points in different colors, the average from each experiment, and the mean of the averages ± SD. **** p < 0.0001, ordinary one-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: Nucleic acid containing protein adducts were quantitated, slot-blotted and TOP3A-DPCs were detected with rabbit polyclonal anti-TOP3A antibody (dilution 1:1000, Proteintech, Rosemont, IL, CAT#: 14525-1-AP).

Techniques: Inhibition, Transfection, Expressing, Quantitation Assay, Comparison, Immunoprecipitation, Plasmid Preparation, Solvent, Control, Magnetic Beads, Western Blot

Journal: Cell reports

Article Title: PARP1-driven repair of topoisomerase IIIα DNA-protein crosslinks by FEN1

doi: 10.1016/j.celrep.2024.114522

Figure Lengend Snippet: (A) Scheme of the modified RADAR assay including FLAG IP for immunodetection of TOP3A-DPCs and their PARylation using anti-TOP3A and anti-PAR antibody. (B) HyperPARylation stabilizes TOP3A-DPCs. RADAR assay samples were prepared from mock-transfected (EV) HCT116 cells or HCT116 cells transfected with FLAG-tagged TOP3A-WT or TOP3A-R364W plasmid constructs for 48 h in the absence or presence of PARGi (1 μM, 1 h pretreatment). Equal amounts (20 μg DNA) of RADAR assay samples were subjected to FLAG IP, digested with benzonase nuclease, run on SDS-PAGE, and immunoblotted with anti-PAR and anti-TOP3A antibodies. EV, empty vector transfected. Equal DNA loading was tested with an anti-dsDNA antibody. (C) PARylation of recombinant TOP3A protein by recombinant PARP1 protein. Following 20 min incubation at room temperature, samples were subjected to IP of TOP3A, followed by western blotting with anti-PAR and anti-TOP3A antibodies. (D) Following 48 h transfection of FLAG-tagged TOP3A-WT, FLAG-tagged TOP3A-R364W expression, or EV, HCT116 cells were pretreated with 1 μM PARGi for 1 h, followed by chromatin fractionation under denaturing conditions and IP with anti-FLAG antibody. Cells expressing TOP3A-R364W were also pretreated with siRNA against TOP3A for 24 h. The immunoprecipitates and input samples were probed with the indicated antibodies. (E) Immunoblotting of FLAG immunoprecipitates from U2OS cells expressing TOP3A-R364W in the presence of PARGi (1 μM, 1 h). Cells were lysed under denaturing conditions and chromatin fractions were subjected to IP either in siControl conditions or siRNA against TOP3A (siTOP3A), PARP1 (siPARP1), and PARP-inhibited (1 μM, 1 h) conditions. The immunoprecipitates and input samples were probed with the indicated antibodies. (F) TOP3A PARylation does not directly affect TOP3A cleavage or reversal activities. Top: Cy3-labeled gapped single-stranded DNA (ssDNA) substrate used for TOP3A activity assay. Bottom: representative gel of TOP3A activity assay. Cleavage sites are indicated by arrows. Reaction products, cleavage, and reversal were generated by TOP3A with the DNA substrate alone, unmodified TOP3A (no PAR), and PARylated TOP3A. Purified TOP3A core (1–637 aa)-RMI1 (1–216 aa) was used in this experiment.

Article Snippet: Nucleic acid containing protein adducts were quantitated, slot-blotted and TOP3A-DPCs were detected with rabbit polyclonal anti-TOP3A antibody (dilution 1:1000, Proteintech, Rosemont, IL, CAT#: 14525-1-AP).

Techniques: Modification, Immunodetection, Transfection, Plasmid Preparation, Construct, SDS Page, Recombinant, Incubation, Western Blot, Expressing, Fractionation, Labeling, Activity Assay, Generated, Purification

Journal: Cell reports

Article Title: PARP1-driven repair of topoisomerase IIIα DNA-protein crosslinks by FEN1

doi: 10.1016/j.celrep.2024.114522

Figure Lengend Snippet: (A) Top: outline of the experimental protocol. TOP3A-R364W-transfected HCT116 cells were pretreated with either DMSO (drug solvent), PARPi (1 μM, 1 h), or PARGi (1 μM, 1 h) before harvest and RADAR assays. Left: representative slot blot probed with anti-TOP3A antibody. Right: quantitation of three independent experiments as shown on the left. Error bar represents mean ± SD. Ordinary one-way ANOVA with Dunnett’s multiple comparison test. DMSO was used as a control condition. ** p < 0.01 (control vs. PARPi) and ns: not significant (control vs. PARGi). (B) Epistasis of FEN1 and PARP1. HCT116 cells were transfected with TOP3A-R364W plasmid constructs for 48 h and co-transfected with either siControl or siFEN1 for 72 h. Before harvest, cells were treated with either the PARPi talazoparib (1 μM, 1h) or DMSO (drug solvent control). TOP3A-DPCs were isolated by RADAR assay and slot blotted with anti-TOP3A antibody. Representative slot blots are shown (left). Right: quantitation of TOP3A-DPCs. Error bars represent data mean ± SD ( n = 3 independent experiments). p values were obtained from ordinary one-way ANOVA with Dunnett’s multiple comparison test. * p < 0.05 (siControl vs. siFEN1), ** p < 0.01 (siControl vs. PARPi), *** p < 0.001 (siControl vs. siFEN1 + PARPi), and ns: not significant (PARPi vs. siFEN1 + PARPi). (C) Left: representative slot blot demonstrating the epistasis of FEN1 and PARP1 for TOP3A-DPC repair. HCT116 cells were transfected with TOP3A-R364W for 48 h. Before harvest, cells were treated with either DMSO (drug solvent) or the PARPi talazoparib (1 μM, 1h), FEN1i (10 μM, 1 h), and a combination of PARPi and FENi. Right: quantitation of the 3 independent experiments as shown on the left. Error bars represent mean ± SD. p values were obtained from ordinary one-way ANOVA with Dunnett’s multiple comparison test. ** p < 0.01 (control vs. FEN1i), *** p < 0.01 (control vs. PARPi), *** p < 0.01 (control vs. FEN1i + PARPi), and ns: not significant (PARPi vs. FEN1i + PARPi). DMSO was used a control. (D) Left: representative confocal microscopy images of γH2AX immunostaining of U2OS cells transfected as indicated. Scale bar, 10 μm. DMSO (drug solvent) was used in the control samples. Right: quantification plot showing fluorescence intensities of γH2AX signal per nucleus (analyzed by ImageJ). Data are the mean ± SD ( n = 100 cells for each condition). Two-tailed unpaired t test with Welch’s correction. *** p < 0.001 (control vs. FENi), *** p < 0.001 (control vs. PARPi), *** p < 0.001 (control vs. FEN1i + PARPi), and ns: not significant (PARPi vs. FEN1i + PARPi). (E) Left: representative images of colony formation assays in HCT116 cells transfected with TOP3A-R364W. Right: quantification of the data from experiments as shown on the left. Clonogenic survival histogram data of each condition are presented after normalization with their respective colony formation efficiency in NT condition. Error bars represent data mean ± SD ( n = 3 independent experiments). Ordinary one-way ANOVA with Dunnett’s multiple comparison test. **** p < 0.0001 (control vs. FENi), **** p < 0.0001 (control vs. PARPi), **** p < 0.0001 (control vs. FENi + PARPi), and ns: not significant (PARPi vs. FEN1i + PARPi). (F) Micronucleus frequency histograms under the condition indicated in (E). Error bar represents data mean ± SD ( n = 3 independent experiments). p values were determined by ordinary one-way ANOVA with Dunnett’s multiple comparison test. *** p < 0.001 (control vs. FENi), *** p < 0.001 (control vs. PARPi), *** p < 0.001 (control vs. FENi + PARPi), and ns: not significant (PARPi vs. FEN1i + PARPi).

Article Snippet: Nucleic acid containing protein adducts were quantitated, slot-blotted and TOP3A-DPCs were detected with rabbit polyclonal anti-TOP3A antibody (dilution 1:1000, Proteintech, Rosemont, IL, CAT#: 14525-1-AP).

Techniques: Transfection, Solvent, Dot Blot, Quantitation Assay, Comparison, Control, Plasmid Preparation, Construct, Isolation, Confocal Microscopy, Immunostaining, Fluorescence, Two Tailed Test

Journal: Cell reports

Article Title: PARP1-driven repair of topoisomerase IIIα DNA-protein crosslinks by FEN1

doi: 10.1016/j.celrep.2024.114522

Figure Lengend Snippet: (A) Representative modified RADAR assay including FLAG IP using the protocol outlined in and showing that TOP3A-DPC ubiquitylation is enhanced by PARPi and inhibited by the ubiquitin (Ub)-activating enzyme E1 inhibitor (UAE1i). Following 48 h transfection with FLAG-tagged TOP3A-R364W, HCT116 cells were treated with the UAE1i (TAK243; 1 μM, 1 h), PARGi (10 μM, 1 h), PARPi (talazoparib; 1 μM, 1 h), PARGi + UAE1i, or PARPi + UAE1i before harvest and RADAR assay. Equal amounts (20 μg DNA) of RADAR assay samples were subjected to IP with FLAG antibody, digested with benzonase nuclease, run on SDS-PAGE, and immunoblotted to detect TOP3Accs with anti-FLAG antibody. The PARylation and ubiquitylation of TOP3Accs were detected with anti-PAR and anti-Ub antibodies. 4%–12% and 8% SDS-PAGE gels were used for PAR and Ub detection. (B) PARP inhibition induces SPRTN interaction with TOP3A-DPCs. U2OS cells expressing FLAG-tagged TOP3A-R364W after 48 h transfection were pretreated with either DMSO (drug solvent), the PARPi talazoparib (1 μM, 1 h), or UAE1i (TAK243; 1 μM, 1 h) alone and a combination of them (PARPi + TAK243) before cell fixation and PLA. Representative images of a PLA for FLAG-tagged TOP3A and endogenous SPRTN. Scale bars, 5 μm. DMSO was used in the control samples. (C) Quantitation of the PLA foci shown in (B). Results are represented as SuperPlots showing individual data points in different colors, the average from each experiment, and the mean of the averages ± SD. p values were obtained by ordinary one-way ANOVA with Tukey’s multiple comparisons test. **** p < 0.0001 (control vs. PARPi), **** p < 0.0001 (control vs. TAK243), and **** p < 0.0001 (PARPi vs. PARPi + TAK243). (D) FEN1 inhibition induces SPRTN interaction with TOP3A-DPCs. U2OS cells transfected with FLAG-tagged TOP3A-R364W were pretreated with either DMSO (drug solvent) or FEN1i (10 μM, 1 h) before cell fixation and PLA. Representative images of a PLA for FLAG-tagged TOP3A and endogenous SPRTN. Scale bars, 5 μm. DMSO was used in the control samples. (E) Quantitation of PLA foci shown in (D). Results are represented as SuperPlots as described in (C). p values were determined by two-tailed unpaired t test with Welch’s correction. **** p < 0.0001.

Article Snippet: Nucleic acid containing protein adducts were quantitated, slot-blotted and TOP3A-DPCs were detected with rabbit polyclonal anti-TOP3A antibody (dilution 1:1000, Proteintech, Rosemont, IL, CAT#: 14525-1-AP).

Techniques: Modification, Ubiquitin Proteomics, Transfection, SDS Page, Inhibition, Expressing, Solvent, Control, Quantitation Assay, Two Tailed Test

Journal: Cell reports

Article Title: PARP1-driven repair of topoisomerase IIIα DNA-protein crosslinks by FEN1

doi: 10.1016/j.celrep.2024.114522

Figure Lengend Snippet: (A) Representative slot blots of TOP3A-DPCs detected using anti-TOP3A antibody (top). HCT116 cells were transfected with TOP3A-R364W for 48 h. Before harvest, cells were treated with either DMSO (drug solvent), the UAE1i TAK243 (1 μM, 2 h), or the proteasome inhibitor bortezomib (1 μM, 2 h). The bottom shows the quantification of TOP3A-DPCs from 3 independent experiments. Error bars indicate mean ± SD. DMSO was used as a control. p values were obtained by ordinary one-way ANOVA with Dunnett’s multiple comparison test. ** p < 0.01 (control vs. TAK243) and ns: not significant (control vs. BTZ). (B) Epistasis of Ub signaling and SPRTN. HCT116 cells were transfected with either siControl or siFEN1 for 72 and 24 h after co-transfection with TOP3A-R364W for 48 h before performing RADAR assays for TOP3A-DPC detection. Before harvest, cells were treated with either DMSO (drug solvent control) or the UAEi TAK243 (1 μM, 1 h). A representative slot blot is shown (top) with quantitation of TOP3A-DPCs (bottom) from three independent experiments. Error bars indicate mean ± SD. p values were obtained by ordinary one-way ANOVA with Dunnett’s multiple comparison test. **** p < 0.001 (siControl vs. siSPRTN), ** p < 0.01 (siControl vs. siControl + TAK243), **** p < 0.0001 (siControl vs. siSPRTN + TAK243), and ns: not significant (siSPRTN vs. siSPRTN + TAK243). (C) Additive effects of FEN1 and SPRTN depletion on TOP3A-DPCs. HCT116 cells were transfected with siFEN1, siSPRTN, and a combination of siFEN1 and siSPRTN for 72 h and, 24 h later, co-transfected with TOP3A-R364W for 48 h before performing RADAR assays. A representative slot blot is shown (top) with quantitation of TOP3A-DPCs from 3 independent experiments (bottom). Error bars indicate mean ± SD. p values were obtained by ordinary one-way ANOVA with Dunnett’s multiple comparison test. * p < 0.05 (siControl vs. siFEN1), **** p < 0.0001 (siControl vs. siSPRTN), **** p < 0.0001 (siControl vs. siSPRTN/siFEN1), and **** p < 0.0001 (siSPRTN vs. siSPRTN/siFEN1). (D) Top: representative slot blot showing the additive effects of FEN1 inhibition and SPRTN depletion on TOP3A-DPCs. HCT116 cells were transfected with siSPRTN for 72 h or siControl. Before harvest, cells were treated with either FEN1i (10 μM, 1 h) or DMSO (solvent control), followed by RADAR assay to measure TOP3A-DPCs by slot blot. Bottom: quantitation of 3 independent experiments as shown on the left. Error bars indicate mean ± SD. p values were obtained by ordinary one-way ANOVA with Dunnett’s multiple comparisons test. ** p < 0.01 (siControl vs. siSPRTN), ** p < 0.01 (siControl vs. siControl + FEN1i), *** p < 0.001 (siControl vs. siSPRTN + FEN1i), and *** p < 0.001 (siSPRTN vs. siSPRTN + FEN1i). (E) Left: representative images of γH2AX immunostaining of TOP3A-R364W-transfected U2OS cells as indicated. Scale bar, 10 μm. Right: quantification plot showing fluorescence intensities of γH2AX signal per nucleus (analyzed by ImageJ). Data are the mean ± SD ( n = at least 100 cells for each condition). Two-tailed unpaired t test with Welch’s correction. **** p < 0.001 (siControl vs. siFEN1), **** p < 0.0001 (control vs. siSPRTN), **** p < 0.0001 (siControl vs. siFEN1/siSPRTN), and **** p < 0.0001 (siSPRTN vs. siFEN1/siSPRTN). (F) Top: representative images of colony formation assays in indicated conditions of HCT116 cells after transfection with TOP3A-R364W. Right: quantification of the data from experiments as shown on the left. Clonogenic survival histogram data of each condition are presented after being normalized with their respective colony formation efficiency in NT conditions. Error bars represent mean ± SD ( n = 3 independent experiments). Ordinary one-way ANOVA with Dunnett’s multiple comparisons test. **** p < 0.001 (siControl vs. siFEN1), **** p < 0.001 (control vs. siSPRTN), **** p < 0.0001 (siControl vs. siFEN1/siSPRTN), and ** p < 0.01 (siSPRTN vs. siFEN1/siSPRTN).

Article Snippet: Nucleic acid containing protein adducts were quantitated, slot-blotted and TOP3A-DPCs were detected with rabbit polyclonal anti-TOP3A antibody (dilution 1:1000, Proteintech, Rosemont, IL, CAT#: 14525-1-AP).

Techniques: Transfection, Solvent, Control, Comparison, Cotransfection, Dot Blot, Quantitation Assay, Inhibition, Immunostaining, Fluorescence, Two Tailed Test

Journal: Cell reports

Article Title: PARP1-driven repair of topoisomerase IIIα DNA-protein crosslinks by FEN1

doi: 10.1016/j.celrep.2024.114522

Figure Lengend Snippet: (A) Additive effects of FEN1 and MRE11 depletion on TOP3A-DPCs. U2OS cells were transfected with siFEN1, siMRE11, and a combination of siFEN1 and siMRE11 for 72 h and, 24 h later, co-transfected with TOP3A-R364W for 48 h before performing RADAR assays. A representative slot blot is shown (top) with quantitation of TOP3A-DPCs from 3 independent experiments (bottom). Error bars indicate mean ± SD. p values were obtained by ordinary one-way ANOVA with Dunnett’s multiple comparisons test. ** p < 0.01 (siControl vs. siFEN1), *** p < 0.001 (siControl vs. siMRE11), **** p < 0.0001 (siControl vs. siFEN1/siMRE11), and *** p < 0.001 (siMRE11 vs. siFEN1/siMRE11). (B) Additive effects of FEN1 depletion and ATM inhibition on TOP3A-DPCs. U2OS cells were transfected with TOP3A-R364W plasmid for 48 h. Cells were co-transfected with either siControl or siFEN1 for 72 h. Before harvest and RADAR assays, cells were treated with either DMSO or the ATMi KU-55933 (20 μM) for 2 h. A representative slot blot is shown (top) with quantitation of TOP3A-DPCs from 3 independent experiments (bottom). Error bars indicate mean ± SD. p values were obtained by ordinary one-way ANOVA with Dunnett’s multiple comparisons test. ** p < 0.01 (siControl vs. siFEN1), ** p < 0.01 (siControl vs. ATMi), *** p < 0.001 (siControl vs. siFEN1 + ATMi), and ** p < 0.01 (siFEN1 vs. siFEN1 + ATMi). (C) Differential roles of FEN1 and MRE11/ATM for TOP3A-DPC repair as a function of cell cycle. U2OS cells were transfected with either siControl or siFEN1, siMRE11 followed by transfection with TOP3A-R364W. siControl cells were treated with the ATMi KU-55933 (20 μM) for 2 h before harvest. Cells (in S and G2/M phases) were collected after synchronization by double-thymidine block. RADAR assays were performed with anti-TOP3A antibody. Representative slot blot images are shown (top). Quantitation from 3 independent RADAR assays is shown on the bottom. Error bars indicate mean ± SD. p values were obtained by two-way ANOVA with Tukey’s multiple comparisons test. **** p < 0.0001 and ns: not significant. (D) Two types of post-translational modifications, PARylation and ubiquitylation, direct the processing of trapped TOP3Accs (TOP3A-DPCs). Left: PARylation of TOP3A-DPCs recruits FEN1. Right: following dePARylation by PARG, ubiquitylation recruits SPRTN for proteolytic degradation of TOP3A-DPCs.

Article Snippet: Nucleic acid containing protein adducts were quantitated, slot-blotted and TOP3A-DPCs were detected with rabbit polyclonal anti-TOP3A antibody (dilution 1:1000, Proteintech, Rosemont, IL, CAT#: 14525-1-AP).

Techniques: Transfection, Dot Blot, Quantitation Assay, Inhibition, Plasmid Preparation, Blocking Assay

Journal: Cell reports

Article Title: PARP1-driven repair of topoisomerase IIIα DNA-protein crosslinks by FEN1

doi: 10.1016/j.celrep.2024.114522

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Nucleic acid containing protein adducts were quantitated, slot-blotted and TOP3A-DPCs were detected with rabbit polyclonal anti-TOP3A antibody (dilution 1:1000, Proteintech, Rosemont, IL, CAT#: 14525-1-AP).

Techniques: Virus, Recombinant, Modification, Protease Inhibitor, Transfection, Magnetic Beads, In Situ, Mutagenesis, Imaging, Control, Cloning, Software