slx4  (Bethyl)

Journal: Nature Communications

Article Title: The CIP2A-TOPBP1 axis facilitates mitotic DNA repair via MiDAS and MMEJ

doi: 10.1038/s41467-025-65594-2

Figure Lengend Snippet: a eGFP-TOPBP1 domain architecture schematic depicting relative positions of TOPBP1 BRCA1 C-terminal (BRCT) domains 0–8 and its ATR activation domain (AAD). b Western blot analysis of BLM, TOP3A, SLX4, MUS81 and ERCC1 in eGFP-TOPBP1 truncation mutant Co-IPs from HEK293TN cells transiently transfected with WT or C-terminally truncated eGFP-TOPBP1 expression constructs followed by 18 h 100 ng/ml nocodazole synchronisation from Supplementary Fig. . Mock Co-IP from non-transfected HEK293TN cells was conducted as negative control (CTRL). c Western blot analysis of SLX4, MUS81 and ERCC1 in eGFP-TOPBP1 Co-IPs from HEK293TN cells transiently transfected with WT or N-terminally truncated ∆314 (3–8) eGFP-TOPBP1 expression constructs followed by 18 h 100 ng/ml nocodazole synchronisation. Mock Co-IP from non-transfected HEK293TN cells was conducted as negative control (CTRL). For all IP experiments 1% of input was used for analysis by western blot of input lysate. d Domain architecture of SLX4 depicting relative positions of the MUS312/MEI9 interaction-like (MLR), Broad-complex, Tramtrack, and Bric-a-brac (BTB), SAF-A/B, Acinus, and PIAS (SAP) and Conserved C-terminal Domain (CCD) domains and the relative position of the identified pT1260, TOPBP1 BRCT 1 recognition motif. e Fluorescence polarisation analysis of recombinant TOPBP1 BRCT0-1-2, BRCT 4-5 and BRCT 7-8 domain containing fragments in the presence of a fluorescein tagged SLX4 pT1260-containing peptide. f Fluorescence polarisation analysis of recombinant TOPBP1 BRCT0-1-2 fragment in the presence of fluorescein tagged SLX4 pT1260-containing peptide with or without lambda (λ) phosphatase treatment. g Fluorescence polarisation analysis of recombinant TOPBP1 BRCT0-1-2 WT or conserved lysine ‘K’ to glutamic acid ‘E’ mutations in the BRCT1 (K155E) or 2 (K250E) or in BRCT 1 + 2 (K155E + K250E) of the protein fragment, in the presence of fluorescein tagged SLX4 pT1260 containing peptide ( e–g contains data from three independent experiments, error bars display SEM, for all FP experiments raw data was fit with a specific and non-specific component, with the non-specific component subtracted before plotting). h Table showing the dissociation constant of TOPBP1 BRCT 0-1-2 WT or K250E with SLX4 pT1260. i TOPBP1 BRCT 0-1-2 and SLX4 pT260 interaction modelled in AlphaFold 3. j TOPBP1 BRCT 0-1-2 and RAD9 pS387 crystal structure (PDB: 6HM5). Source data are provided as a file.

Article Snippet: The SLX4 coding sequence was transferred to pcDNA5-Neo-FRT/TR (RRID: Addgene_41000; a gift from Dr. J. Mansfeld) via HiFi DNA assembly following manufacturer’s guidelines (NEB, E2621S). pcDNA5-Neo-FRT/TO-eGFP-SLX4 T1260A plasmid was generated by subjecting the pcDNA5-FRT/TO-Neo-eGFP-SLX4 plasmid to site directed mutagenesis using the Q5 site directed mutagenesis kit (NEB, E0552S). pENTR223.1-POLQ (HsCD00353909 ) was obtained from the DNASU plasmid repository.

Techniques: Activation Assay, Western Blot, Mutagenesis, Transfection, Expressing, Construct, Co-Immunoprecipitation Assay, Negative Control, Fluorescence, Recombinant

Journal: Nature Communications

Article Title: The CIP2A-TOPBP1 axis facilitates mitotic DNA repair via MiDAS and MMEJ

doi: 10.1038/s41467-025-65594-2

Figure Lengend Snippet: a Western blot analysis of fractionated HEK293TN cells treated with 100 ng/ml of nocodazole for 18 hours, 24 hours after transient transfection with eGFP-TOPBP1 WT, or 1 + 2 (K155E + K250E) expression constructs and western blot analysis of SLX4, MUS81 and ERCC1 in Co-IPs from the chromatin fraction of these cells. Mock Co-IP from non-transfected HEK293TN cells was conducted as negative control (CTRL). b Western blot analysis of TOPBP1, MUS81 and ERCC1 in eGFP-SLX4 Co-IPs from the chromatin fraction of HEK293TN cells treated with 100 ng/ml of nocodazole for 18 h after transient transfection of eGFP-SLX4 WT and T1260A expression constructs. Mock Co-IP from non-transfected HEK293TN cells was conducted as negative control (CTRL). c Western blot analysis of CDK1 dependent phosphorylation of SLX4 T1260 and interaction with MUS81, ERCC1 and TOPBP1 in eGFP-SLX4 Co-IPs. HEK293TN cells were transfected with an eGFP-SLX4 expression construct and 24 h later were synchronised for 18 h with 100 ng/ml nocodazole. This was followed with or without 7 µM RO-3306 (CDK1i) treatment for 30 min, prior to collection. Mock Co-IP from non-transfected HEK293TN cells act as negative control (CTRL). d As in C, but reciprocal eGFP-TOPBP1 Co-IP analysis. For all IP experiments 1% of input was used for analysis by western blot of input lysate. e SDS-PAGE of in vitro reconstitution of CDK1-Cyclin B-CKS1 driven phosphorylation of a SLX4 T1260 containing biotinylated peptide and its phosphorylation-dependent interaction with a recombinant TOPBP1 BRCT 0-1-2 containing fragment, demonstrated through peptide pull down. Source data are provided as a file.

Article Snippet: The SLX4 coding sequence was transferred to pcDNA5-Neo-FRT/TR (RRID: Addgene_41000; a gift from Dr. J. Mansfeld) via HiFi DNA assembly following manufacturer’s guidelines (NEB, E2621S). pcDNA5-Neo-FRT/TO-eGFP-SLX4 T1260A plasmid was generated by subjecting the pcDNA5-FRT/TO-Neo-eGFP-SLX4 plasmid to site directed mutagenesis using the Q5 site directed mutagenesis kit (NEB, E0552S). pENTR223.1-POLQ (HsCD00353909 ) was obtained from the DNASU plasmid repository.

Techniques: Western Blot, Transfection, Expressing, Construct, Co-Immunoprecipitation Assay, Negative Control, Phospho-proteomics, SDS Page, In Vitro, Recombinant

Journal: Nature Communications

Article Title: The CIP2A-TOPBP1 axis facilitates mitotic DNA repair via MiDAS and MMEJ

doi: 10.1038/s41467-025-65594-2

Figure Lengend Snippet: a Representative images, a bar plot showing the mean percentage of eGFP-SLX4 WT foci colocalising with CIP2A per RPE1 p53 -/- SLX4 -/- FRT/TR +eGFP-SLX4 WT/ T1260A prometaphase cell after induction with 10 ng/ml doxycycline for 24 h followed by treatment with or without 400 nM/ 18 h aphidicolin followed by synchronisation with 60 ng/ml nocodazole for 2 h (UT: n = 85, Aph: n = 76), and a dot plot showing the number of eGFP-SLX4 WT or eGFP-SLX4 T1260A foci colocalising with CIP2A foci per prometaphase cell after treatment as above (eGFP-SLX4 WT (UT: n = 85, Aph: n = 76) and eGFP-SLX4 T1260A (UT: n = 70, Aph: n = 76)) from three independent experiments, statistical significance was determined by two-way ANOVA). Grey dots represent individual measurements, black dots represent the medians of each experiment and bars show the mean with error bars showing SEM. b Representative images and a dot plot showing CIP2A and MUS81 colocalising foci in RPE1 p53 -/- FRT/TR WT and SLX4 T1260A knock in prometaphase cells treated with 400 nM aphidicolin followed by synchronisation with 60 ng/ml nocodazole for 2 h (Parental WT: n = 80, T1260A Cl.1: n = 76, T1260A Cl.2 n = 85, from three independent experiments, statistical significance was determined by one-way ANOVA test). Grey dots represent individual measurements, black dots represent the medians of each experiment and bars show the mean with error bars showing SEM. c Representative images and violin plot of the number of EdU foci in RPE1 p53 -/- FRT/TR WT and T1260A prometaphase cells (Parental WT: n = 90, T1260A Cl.1: n = 87, T1260A Cl.2 n = 82, from three independent experiments, statistical significance was determined by two-sided Mann-Whitney U test). d Representative images and bar plot of the mean of the percentage of micronuclei-positive RPE1 p53 -/- FRT/TR WT, SLX4 -/ - and SLX4 T1260A knock in cells (Parental WT n = 336, SLX4 -/- n = 346, T1260A Cl.1: n = 384, T1260A Cl.2 n = 377 from three independent experiments, statistical significance was determined by one-way ANOVA), black dots represent the mean of each experiment and bars show the mean with error bars showing SEM. Scale bars in ( a–d ) are equivalent to 10 µm. Source data are provided as a file.

Article Snippet: The SLX4 coding sequence was transferred to pcDNA5-Neo-FRT/TR (RRID: Addgene_41000; a gift from Dr. J. Mansfeld) via HiFi DNA assembly following manufacturer’s guidelines (NEB, E2621S). pcDNA5-Neo-FRT/TO-eGFP-SLX4 T1260A plasmid was generated by subjecting the pcDNA5-FRT/TO-Neo-eGFP-SLX4 plasmid to site directed mutagenesis using the Q5 site directed mutagenesis kit (NEB, E0552S). pENTR223.1-POLQ (HsCD00353909 ) was obtained from the DNASU plasmid repository.

Techniques: Knock-In, MANN-WHITNEY

Journal: Nature Communications

Article Title: The CIP2A-TOPBP1 axis facilitates mitotic DNA repair via MiDAS and MMEJ

doi: 10.1038/s41467-025-65594-2

Figure Lengend Snippet: a Representative images and bar plot illustrating the number of micronuclei per RPE1 p53 -/- FRT/TR WT or SLX4 T1260A knock in cell nucleus. Cells were treated with siCTRL or siCIP2A, 400 nM aphidicolin, in addition to either DMSO or 5 µM ART558 (Polθi) for 18 h (Parental WT (siCTRL + DMSO: n = 343, siCIP2A + DMSO: n = 394, siCTRL + Polθi: n = 372), SLX4 T1260A Cl.1 (siCTRL + DMSO: n = 385 and siCTRL + Polθi: n = 341) or SLX4 T1260A Cl.2 (siCTRL + DMSO: n = 399 and siCTRL + Polθi: n = 378) from three independent experiments). Statistical significance was determined using by one-way ANOVA, and the mean of each experiment is displayed as black dots and the mean of all three experiments is presented as bars with SEM. Scale bars are equivalent to 10 µm. b Proliferation analysis of RPE1 p53 -/- FRT/TR WT and T1260A cells, performed using the Incucyte live cell analysis system. Cells were treated with DMSO or with 5 µM ART558 (Polθi) in the presence of 400 nM aphidicolin. The mean confluence divided by confluence at day 0, from three independent experiments, is displayed with SEM. c Proliferation analysis of DLD1 BRCA2 -/- cells using the Incucyte live cell analysis system. Cells were treated with siCTRL, siSLX4 or siCIP2A in the presence of DMSO or 5 µM ART558 (Polθi). The mean confluence divided by confluence at day 0, from three independent experiments, is displayed with SEM. d Model: Cells enter mitosis with DNA damage, under replicated DNA or recombination intermediates where the CIP2A-TOPBP1 complex is recruited. CDK1 phosphorylation of SLX4 at T1260 regulates interaction with TOPBP1 BRCT 1/2 facilitating SMX component recruitment and MiDAS/BIR to safeguard genome stability. PLK1 phosphorylation of POLQ in mitosis facilitates TOPBP1 interaction and MMEJ . Loss of CIP2A impairs both pathways and leads to simultaneous deficiency in mitotic MiDAS/BIR and MMEJ promoting genome instability. Created in BioRender. Martin, P. (2025) https://BioRender.com/d5kygf2 . Source data are provided as a Source Data file.

Article Snippet: The SLX4 coding sequence was transferred to pcDNA5-Neo-FRT/TR (RRID: Addgene_41000; a gift from Dr. J. Mansfeld) via HiFi DNA assembly following manufacturer’s guidelines (NEB, E2621S). pcDNA5-Neo-FRT/TO-eGFP-SLX4 T1260A plasmid was generated by subjecting the pcDNA5-FRT/TO-Neo-eGFP-SLX4 plasmid to site directed mutagenesis using the Q5 site directed mutagenesis kit (NEB, E0552S). pENTR223.1-POLQ (HsCD00353909 ) was obtained from the DNASU plasmid repository.

Techniques: Knock-In, Cell Analysis, Phospho-proteomics

Journal: bioRxiv

Article Title: The BRCA1- RAD51 Axis Regulates SCAI/REV3 Dependent Replication Fork Maintenance

doi: 10.1101/2025.11.25.689574

Figure Lengend Snippet: A. WT RPE1 cells were transfected and treated with 2 mM HU for 18 h. WCLs were prepared, and WB were performed against the indicated proteins. B. Western blots from WT RPE cells transfected with the indicated siRNAs and then treated with 1 mM HU for 18 h. C . Comet assay of hTERT-RPE1 cells treated with 2 mM HU for 40 h. Indicated siRNAs were transfected two days before HU treatment. Red line represents median, three independent experiments, representative experiment shown. Kruskal-Wallis followed by Dunn’s multiple comparison test. ns – not significant (P ≥ 0.05). D. WT RPE1 cells were transfected with the indicated siRNAs, then treated with 1.5 mM HU for 18 h and fractioned prior to blotting for the indicated proteins. SLX4 values are normalized to ORC2 loading control. E. WT RPE1 cells were transfected and then treated with 2 mM HU for 18 h. Samples were processed for chromatin fraction prior to blotting for the indicated proteins. F. WT RPE1 cells were transfected with the indicated siRNAs, then treated with 2 mM HU for 18 h and fractioned prior to blotting for the indicated proteins.

Article Snippet: The antibodies used in this work include the following: Anti-vinculin 1:1000 (Sigma-Aldrich, V9131-.2ML), vinculin (7F9) 1:200 (Santa Cruz Biotechnology, sc-73614), SCAI 1:500 (Abcam, ab124688), Chk1 (2G1D5) 1:1000 (Cell Signaling, 2360S), P-Chk1 (S345) (133D3) Rabbit mAb 1:1000 (Cell Signaling, 2348S), RPA 32 kDa subunit (9H8) 1:200 (Santa Cruz Biotechnology, sc-56770, only used for western blots), RPA32 1:200 (Genetex, GTX70258, only used for IF), Rabbit x-Phospho RPA32 (S4/S8) 1:1000 (Bethyl, A300-245A), H2AX 1:1000 (Bethyl, A300-082A), Anti-phospho-Histone H2A.X (Ser139) 1:1000 (EMD Millipore Corp., 05-636), REV7 1:200 (Santa Cruz Biotechnology, sc-135977), Anti-Exonuclease 1 (EXO1) 1:1000 (Bethyl, A302-640A), ORC2 1:1000 (Abcam, ab68348), BRCA1 1:1000 (EMD Millipore Corp., 07-434), 53BP1 1:1000 (Bethyl, A300-272A), RIF1 1:1000 (Bethyl, A300-569A), CtIP 1:1000 (Bethyl, A300-488A), SmarcAL1 (A-2) 1:200 (Santa Cruz Biotechnology, sc-376377), ZRANB3 1:1000 (Bethyl, A303-033A), FBH1 1:1000 (Abcam, ab58881), RAD51 1:1000 (Abcam, ab63801), MUS81 1:200 (Santa Cruz Biotechnology, sc-53382), SLX4 1:1000 (Bethyl, A302-270A), H3 1:1000 (Cell Signaling, 9715), MRE11 1:1000 (Abcam, ab214), Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP 1:2500 (Invitrogen, 31430), Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP 1:2500 (Invitrogen, 31460), Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 1:300 (Invitrogen, A32731), Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 594 1:300 (Invitrogen, A32742), Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 1:300 (Invitrogen, A32723).

Techniques: Transfection, Western Blot, Single Cell Gel Electrophoresis, Comparison, Control

Journal: Cancer Biology & Therapy

Article Title: SLX1 silencing overcomes Olaparib resistance in metastatic castration-resistant prostate cancer by disrupting SLX4-mediated DNA repair complexes

doi: 10.1080/15384047.2025.2545062

Figure Lengend Snippet: Knockdown of SLX1 reduces the binding of ERCC1-XPF, PLK1, and TOPBP1 to the SLX1-SLX4 complex.

Article Snippet: Equal amounts of protein (1000 μg) were incubated overnight at 4°C with 1 μg of the indicated primary antibody against SLX4 (NBP1–28680, Novus Biologicals).

Techniques: Knockdown, Binding Assay