pcdna3 1 ros1 v5  (Addgene inc)

Journal: bioRxiv

Article Title: CIP2A is required for mitotic recruitment of the SLX1/XPF/MUS81 tri-nuclease complex to replication stress-induced DNA lesions to maintain genome integrity

doi: 10.1101/2025.04.03.647079

Figure Lengend Snippet: (A) RPE1 TP53 -/- cells were either left untreated or treated with aphidicolin (APH, 200 nM, 20 h) or ionizing radiation (IR, 0.25 Gy). Cells were stained for DAPI (blue), CIP2A (green) and ψH2AX (red). Scale bar represents 10 µm. (B) Quantification of co-localizing CIP2A and ψH2AX foci per mitotic cell for cells as treated as described in A. Individual values and medians of n>30 cells per condition are shown. (C) RPE1 TP53 -/- cells and CIP2A -/- clones were either left untreated or treated with APH (200 nM, 24 h) or IR (3 Gy) and analyzed 72 h later. Micronuclei per cell were quantified. The bars represent the mean and standard error of the mean (SEM) from three biologically independent experiments with n≥64 cells per experimental condition. P-values were calculated using two-tailed unpaired t-test. (D, E) RPE1 TP53 -/- cells were treated with APH (200 nM, 20 h, panel D) or IR (0.25 Gy, panel E). Representative images of three classes of mitotic structures are shown. Left panels show the confocal overview images, and the right three panels show STED images of a single CIP2A-TOPBP1 complex. Cells were stained for DAPI (blue), CIP2A (green) and TOPBP1 (red). Scale bar represents 5 µm (confocal) or 500 nm (STED). STED images are Wiener deconvolved. Raw data is shown in Suppl. Fig 1G, H. (F) Quantification of CIP2A-TOPBP1 structures per cell per mitotic phase for cells treated as described in panel D. Number of cells quantified per mitotic phase from three biologically independent experiments: prophase (n=9), prometaphase (n=9), metaphase (n=19), anaphase (n=18), telophase (n=29). The numbers of CIP2A-TOPBP1 structures per telophase may be an overestimation of the actual number, as only cells with CIP2A-TOPBP1 structures were measured. Bars represent mean and SEM of three experiments. (G) Quantification of CIP2A-TOPBP1 structures per cell per mitotic phase for cells treated with IR as described in panel E. Number of cells quantified per mitotic phase from three biologically independent experiments: prophase (n=11), prometaphase (n=10), metaphase (n=14), anaphase (n=13), telophase (n=19). Bars represent mean and SEM of three biologically independent experiments. (H) Quantification of unstructured, loop-containing and filamentous structures per mitotic phase for indicated treatments, as observed with STED microscopy for cells treated as described in from panels D and E. Percentages compared to the total structures are indicated. Mean, SEM and n, which reflects the total number of observed structures per phase, from three biologically independent experiments are shown per mitotic phase and per treatment. (I) Quantification of the size of unstructured, loop-containing and filamentous structures per mitotic phase for indicated treatments, as observed with STED microscopy for cells treated as described in panels D and E. Structure size was determined by measuring the length of the longest axis through the entire structure as shown in Suppl. Fig. 1J. Individual values, the average of medians per experiment, along with SEM, are plotted per mitotic phase, ‘n’ represents the total number of observed structures per phase from three biologically independent experiments.

Article Snippet: Full length CIP2A was cloned from pcDNA3.1/CIP2A(1-905) WT V5 His (Addgene #119287), which was a gift from Jukka Westermarck , into retroviral pMSCV-blast which was a gift from David Mu (Addgene #75085) , and subsequently different CIP2A mutations were generated in this pMSCV plasmid.

Techniques: Staining, Clone Assay, Two Tailed Test, Microscopy

Journal: bioRxiv

Article Title: CIP2A is required for mitotic recruitment of the SLX1/XPF/MUS81 tri-nuclease complex to replication stress-induced DNA lesions to maintain genome integrity

doi: 10.1101/2025.04.03.647079

Figure Lengend Snippet: (A, B) Mass spectrometry analysis of mitotic CIP2A interactors in RPE1 TP53 -/- parental versus CIP2A -/- cl#1 cells upon treatment with IR (panel A, 5 Gy) or with APH (panel B, 200 nM, 20 h). Proteins highlighted in red are enriched after IR or APH treatment. (C) Left panel: representative images of RPE1 TP53 -/- cells stained for DAPI (blue), CIP2A (green) and XPF (red) in either untreated conditions, or upon treatment with APH (200 nM, 20 h) or IR (0,25 Gy). Scale bar represents 10 µm. Right panel: quantification of the percentage of CIP2A foci per mitotic cell that co-localize with XPF. Bar represents the median of one experiment with n>35 cells per experimental condition. (D) RPE1 TP53 -/- and CIP2A -/- cl#1 cells were treated with APH (200 nM, 20 h). Representative images of treated cells stained for DAPI (blue), CIP2A (green) and XPF (red). Scale bar represents 10 µm. (E) Representative images of RPE1 TP53 -/- cells and CIP2A -/- cl#1 cells treated with APH (200 nM, 20 h). Cells were stained for DAPI (blue), CIP2A (red) and SLX4 (green). Scale bar represents 10 µm. (F) RPE1 TP53 -/- cells and CIP2A -/- cl#1 cells were left untreated or treated with APH (200 nM, 20 h). Representative images of treated cells stained for DAPI (blue), CIP2A (red) and MUS81 (green) in untreated condition or after treatment with APH (200 nM, 20 h). Scale bar represents 10 µm. (G) RPE1 TP53 -/- cells and CIP2A -/- cl#1 cells were left untreated or treated with APH (200 nM, 20 h). Representative images of treated cells stained for DAPI (blue), CIP2A (red) and ERCC1 (green), in untreated conditions or after treatment with APH (200 nM, 20 h). Scale bar represents 10 µm. (H) Quantification of mitotic XPF foci in RPE1 TP53 -/- cells and CIP2A -/- cl#1 cells either left untreated or treated as described in D. Individual values, medians and interquartile range of three experiments with n>30 cells per experimental condition are plotted. P-values were calculated using two-way ANOVA with Šidák’s multiple comparisons test on median values per experiment. (I) Quantification of mitotic SLX4 foci in RPE1 TP53 -/- cells and CIP2A -/- cl#1 cells either untreated or treated as described in panel E. Individual values, medians and interquartile range of three experiments with n>30 cells per experimental condition are plotted. P-values were calculated using two-way ANOVA with Šidák’s multiple comparisons test on the medians per experiment. (J) Quantification of mitotic MUS81 foci in RPE1 TP53 -/- cells and CIP2A -/- cl#1 cells either left untreated or treated as described in panel F. Individual values, medians and interquartile range of three experiments with n≥27 cells per experiment are plotted. P-values were calculated using two-way ANOVA with Šidák’s multiple comparisons test on the medians per experiment. (K) Quantification of mitotic ERCC1 foci in RPE1 TP53 -/- cells and CIP2A -/- cl#1 cells either left untreated or treated as described in panel G. Individual values, medians and interquartile range of three experiments with n≥28 cells per experimental condition are plotted. P-values were calculated using two-way ANOVA with Šidák’s multiple comparisons test on the medians per experiment.

Article Snippet: Full length CIP2A was cloned from pcDNA3.1/CIP2A(1-905) WT V5 His (Addgene #119287), which was a gift from Jukka Westermarck , into retroviral pMSCV-blast which was a gift from David Mu (Addgene #75085) , and subsequently different CIP2A mutations were generated in this pMSCV plasmid.

Techniques: Mass Spectrometry, Staining

Journal: bioRxiv

Article Title: CIP2A is required for mitotic recruitment of the SLX1/XPF/MUS81 tri-nuclease complex to replication stress-induced DNA lesions to maintain genome integrity

doi: 10.1101/2025.04.03.647079

Figure Lengend Snippet: (A-C) RPE1 TP53 -/- cells were treated with APH (200 nM, 20 h) and stained for DAPI (blue), CIP2A (green) and SLX4 (red, panel A), ERCC1 (red, panel B), MUS81 (red, panel C). A representative image of each of the CIP2A structure organizations (unstructured, loop-containing, filamentous) are shown. Scale bar represents 5 µm (confocal) or 500 nm (STED). STED images are Wiener deconvolved. Raw data is shown in Suppl. Fig. 3A-C. (D-F) Quantification of the percentage of mitotic CIP2A structures that co-localize with SLX4 (panel D), ERCC1 (panel E) or MUS81 (panel F) as observed with STED microscopy for each of the CIP2A structure organizations (unstructured, loop-containing, filamentous) for cells treated as described in panels A-C. Bars represent means and SEM of three experiments with n>75 structures per experiment. (G) RPE1 TP53 -/- cells were treated with APH (200 nM, 20 h) and stained for DAPI (blue), CIP2A (green) and ψH2AX (red). Scale bar represents 5 µm (confocal) or 500 nm (STED). STED images are Wiener deconvolved. Raw data is shown in Suppl. Fig. 3D. (H) RPE1 TP53 -/- cells were treated with APH (200 nM, 20 h), pulsed with EdU during mitotic entry and stained for DAPI (blue), CIP2A (green) and EdU (red). Scale bar represents 5 µm (confocal) or 500 nm (STED). STED images are Wiener deconvolved. Raw data is shown in Suppl. Fig 3E. (I) Quantification of mitotic CIP2A structures that are positive for ψH2AX as observed by STED microscopy for cells treated as described in panel G. Bars represent means and SEM for three experiments with n>75 structures per experiment. (J) Quantification of mitotic CIP2A structures that are either negative (0 foci) or positive for 1, 2 or more than 2 EdU foci as observed by STED microscopy for cells treated as described in panel I. Pie chart represents the means of three experiments with n>75 structures per experiment.

Article Snippet: Full length CIP2A was cloned from pcDNA3.1/CIP2A(1-905) WT V5 His (Addgene #119287), which was a gift from Jukka Westermarck , into retroviral pMSCV-blast which was a gift from David Mu (Addgene #75085) , and subsequently different CIP2A mutations were generated in this pMSCV plasmid.

Techniques: Staining, Microscopy

Journal: bioRxiv

Article Title: CIP2A is required for mitotic recruitment of the SLX1/XPF/MUS81 tri-nuclease complex to replication stress-induced DNA lesions to maintain genome integrity

doi: 10.1101/2025.04.03.647079

Figure Lengend Snippet: (A) RPE1 TP53 -/- , CIP2A -/- cl#1 cells and CIP2A-V5 reconstituted CIP2A -/- cl#1 cells were treated with APH (200 nM, 20 h), pulsed with EdU during mitotic entry and stained for DAPI (blue) and EdU (red) Scare bar represents 10 µm. (B) Quantification of EdU foci per mitotic cell in untreated RPE1 TP53 -/- cells, CIP2A -/- cl#1 cells, and CIP2A -/- cl#1 cells reconstituted with full CIP2A WT-V5 or cells treated as described in panel A. Individual values, medians and interquartile range of three experiments with n>28 cells per experimental condition are plotted. P-values were calculated using ordinary one-way ANOVA with Šidák’s multiple comparisons test on the medians per experiment. (C) Wildtype or CIP2A -/- MDA-MB-231 cells were treated with APH (200 nM, 20 h) and pulsed with EdU during mitotic entry. Representative images of MDA-MB231 cells stained for DAPI, CIP2A, SLX4, EdU are shown. Scale bar represents 10 µm. (D) Quantification of EdU and SLX4 foci per mitotic cell for cells treated as described in panel C. Individual values, medians and interquartile range of three experiments with n≥29 cells per experimental condition are plotted. P-values were calculated using two-tailed unpaired t-test on the medians per experiment. (E) Wildtype or CIP2A -/- HCC38 cells, and doxycycline-treated BT549 cells with doxycycline-inducible scramble (shSCR) or CIP2A (shCIP2A) shRNA were treated with APH (200 nM, 20 h), and pulsed with EdU during mitotic entry. Quantification of EdU foci per mitotic cell in HCC38 and BT549. Individual values, medians and interquartile range of three experiments with n>30 cells per experimental conditions are plotted. P-values are calculated using two-tailed unpaired t-test on the medians per experiment.

Article Snippet: Full length CIP2A was cloned from pcDNA3.1/CIP2A(1-905) WT V5 His (Addgene #119287), which was a gift from Jukka Westermarck , into retroviral pMSCV-blast which was a gift from David Mu (Addgene #75085) , and subsequently different CIP2A mutations were generated in this pMSCV plasmid.

Techniques: Staining, Two Tailed Test, shRNA

Journal: bioRxiv

Article Title: CIP2A is required for mitotic recruitment of the SLX1/XPF/MUS81 tri-nuclease complex to replication stress-induced DNA lesions to maintain genome integrity

doi: 10.1101/2025.04.03.647079

Figure Lengend Snippet: (A) CIP2A is predicted to form a stable homodimer, involving a globular domain with a long coiled-coil. Two predicted binding sites of TOPBP1 are indicated, along with an uncertain, and possibly flexible, position of the N-terminal regions onto the coiled-coil shaft. (B) A cartoon and surface representation of the predicted CIP2A:CIP2A dimer structure with two copies of TOPBP1 755-860, and a magnification of predicted TOPBP1:CIP2A binding interfaces. (C) Schematic representation of full length (1-905) CIP2A (WT), the reported dimerization site (L533), the PLK1 phosphorylation site (S904), the globular domain (1-617), the alpha-helical coiled-coil (618-876), and the unstructured C-terminal domain (877-905). Schematic representation of V5-tagged CIP2A variants: CIP2A lacking the unstructured C-terminal domain (‘CIP2A-ΔC’), CIP2A with only globular domain (‘CIP2A-Glob’), CIP2A lacking the alpha-helical coiled-coil (‘CIP2A-ΔCC’) and CIP2A lacking the globular domain (‘CIP2A-ΔGlob’). (D) Quantification of V5 foci and TOPBP1 foci per mitotic cell in RPE1 TP53 -/- CIP2A -/- cl#1 cells reconstituted with indicated CIP2A-V5 cDNAs, treated with APH (200 nM, 20 h). Individual values, medians and interquartile range of three experiments with n>30 cells per experimental condition are plotted. P-values were calculated using ordinary one-way ANOVA with Šidák’s multiple comparisons test on the medians per experiment. (E) Quantification of micronuclei per cell in parental RPE1 TP53 -/- cells, CIP2A -/- cl#1 cells and CIP2A -/- cl#1 cells reconstituted with indicated CIP2A cDNAs, treated with APH (200 nM, 20 h). Individual values, medians and interquartile range of at least three experiments with n≥85 cells per experimental condition are plotted. P-values were calculated using ordinary one-way ANOVA with Dunnett’s multiple comparison test. (F) CIP2A -/- cells reconstituted with CIP2A-WT, CIP2A-S904A or CIP2A-ΔC were treated with APH (200 nM, 20 h), and stained for DAPI (blue), V5 (red) and SLX4 (green). Scale bar represents 10 µm. (G) Quantification of SLX4 foci per mitotic cell in CIP2A -/- cl#1 reconstituted with indicated mutants for cells treated as described in panel F. Individual values, medians and interquartile range of three experiments with n>30 cells per experimental condition are plotted. P-values were calculated using ordinary one-way ANOVA with Šidák’s multiple comparisons test on the medians per experiment. (H) Quantification of MUS81 foci per mitotic cell in CIP2A -/- cl#1 reconstituted with either CIP2A-WT, CIP2A-S904A or CIP2A-ΔC after treatment with APH (200 nM, 20 h). Individual values, medians and interquartile range of three experiments with n>30 cells per experimental condition are plotted. P-values were calculated using ordinary one-way ANOVA with Šidák’s multiple comparisons test on the medians per experiment. (I) Representative STED microscopy images of DAPI (blue), V5 (green) and TOPBP1 (red) structures in CIP2A -/- cells reconstituted with CIP2A-WT for the three classes of structures (unstructured, loop-containing, and filamentous) after APH treatment (200 nM, 20 h). Scale bar represents 5 µm (confocal) or 500 nm (STED). STED images are Wiener deconvolved. Raw data is shown in Suppl. Fig. 7D (J) Representative STED microscopy images of DAPI (blue), V5 (green) and TOPBP1 (red) structures in CIP2A -/- cells reconstituted with CIP2A-ΔC for the three classes of structures (unstructured, loop-containing, and filamentous) after APH treatment (200 nM, 20 h). Scale bar represents 5 µm (confocal) or 500 nm (STED). STED images are Wiener deconvolved. Raw data is shown in Suppl. Fig. 7E. (K) Quantification of unstructured, loop-containing or loop-like, and filamentous or filament-like structures in CIP2A -/- cells reconstituted with either CIP2A-WT or CIP2A-ΔC. Bars represent the mean and SEM of three experiments with n>15 structures per experimental condition. P-values were calculated using two-tailed unpaired t-test.

Article Snippet: Full length CIP2A was cloned from pcDNA3.1/CIP2A(1-905) WT V5 His (Addgene #119287), which was a gift from Jukka Westermarck , into retroviral pMSCV-blast which was a gift from David Mu (Addgene #75085) , and subsequently different CIP2A mutations were generated in this pMSCV plasmid.

Techniques: Binding Assay, Phospho-proteomics, Comparison, Staining, Microscopy, Two Tailed Test

Journal: bioRxiv

Article Title: CIP2A is required for mitotic recruitment of the SLX1/XPF/MUS81 tri-nuclease complex to replication stress-induced DNA lesions to maintain genome integrity

doi: 10.1101/2025.04.03.647079

Figure Lengend Snippet: (A) Representative images of RPE1 TP53 -/- PAC -/- cells and RPE1 TP53 -/- PAC -/- BRCA1 -/- cells, either left untreated or treated with APH (200 nM, 20 h) and stained for DAPI (blue), CIP2A (red) and SLX4 (green). Scale bar represents 10 µm. (B) Representative images of DLD1 WT cells and DLD1 BRCA2 -/- cells, either left untreated or treated with APH (200 nM, 20 h), and stained for DAPI (blue), CIP2A (red) and SLX4 (green) in. Scale bar represents 10 µm. (C) Quantification of CIP2A and SLX4 foci per mitotic cell for cells treated as described in panel A. Individual values, medians and interquartile range of three experiments with n>30 cells per experimental condition are plotted. P-values were calculated using two-way ANOVA with Šidák’s multiple comparisons test on the medians per experiment. Quantification of CIP2A foci co-localizing with SLX4 for cells treated as described in panel A. Median values per experiment are plotted. Bars represent the mean and SD of three experiments with n>30 cells per experimental condition. (D) Quantification of CIP2A and SLX4 foci per mitotic cell for cells treated as described in panel B. Individual values, medians and interquartile range of three experiments with n>30 cells per experimental condition are plotted. P-values were calculated using two-way ANOVA with Šidák’s multiple comparisons test on the medians per experiment. Quantification of the total number of CIP2A foci that co-localize with SLX4 for cells treated as described in panel B. Median values per experiment are plotted. Bars represent the mean and SD of three experiments with n>30 cells per experimental condition. (E) Representative images of clonogenic survival assays of doxycycline-treated parental RPE1 TP53 -/- and CIP2A -/- cl#1 and CIP2A -/- cl#1 cells, reconstituted with CIP2A-WT or CIP2A-ΔC with doxycycline-inducible luciferase (shLUC) or BRCA2 (shBRCA2) shRNA. (F) Quantification of colony survival for cells treated as described in panel E, normalized to doxycycline-treated shLUC-expressing cell lines. Bars represent mean survival and SD of three experiments. P-values were calculated using ordinary one-way ANOVA with Dunnett’s multiple comparison test. (G-H) Representative images of DLD1 BRCA2 -/- cells transfected with indicated siRNAs and stained for DAPI. Scale bar represents 10 µm. Arrows indicate either micronuclei (panel G), or nucleoplasmic bridges (panel H). (I) Quantification of number of micronuclei per cell for DLD1 WT cells or DLD1 BRCA2 -/- cells, transfected with indicated siRNAs. Bars represent the mean with SEM of 6 experiments with at least n>280 cells per experimental condition. P-values were calculated using two-tailed unpaired t-test. (J) Quantification of number of nucleoplasmic bridges per cell for DLD1 WT cells or DLD1 BRCA2 -/- cells transfected with indicated siRNAs. Bars represent the mean with SEM of 6 experiments with at least n>280 cells per experimental condition. P-values were calculated using two-tailed unpaired t-test.

Article Snippet: Full length CIP2A was cloned from pcDNA3.1/CIP2A(1-905) WT V5 His (Addgene #119287), which was a gift from Jukka Westermarck , into retroviral pMSCV-blast which was a gift from David Mu (Addgene #75085) , and subsequently different CIP2A mutations were generated in this pMSCV plasmid.

Techniques: Staining, Luciferase, shRNA, Expressing, Comparison, Transfection, Two Tailed Test