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

Journal: Nucleic Acids Research

Article Title: SLX4 dampens MutSα-dependent mismatch repair

doi: 10.1093/nar/gkac075

Figure Lengend Snippet: SLX4 interacts with both MutSα and MutSβ through a conserved N-terminal region. ( A ) HeLa FITo cells were transfected with FLAG-HA-SLX4 (FHA-SLX4) vector before FLAG immunoprecipitation (IP) and western blotting, which shows that MSH2 co-immunoprecipitates with overexpressed SLX4. ( B ) IP of endogenous SLX4. Co-immunoprecipitation (coIP) of MSH2 with endogenous SLX4 is barely detectable compared to experiments using overexpressed FHA-SLX4 as in A. The asterisk indicates an aspecific band recognized by the anti-SLX4 antibody ( C ) Left panel: IP of endogenous SLX4 from a nuclear soluble fraction or a chromatin-solubilized fraction in which the coIP of MSH2, MSH3 and MSH6 is readily detected. Right panel: control of the fractionation by western blot against CHK1, mainly nuclear soluble, and SMC3 enriched in chromatin. ( D ) Scheme of SLX4 illustrating the location and conservation of a short domain representing the putative MSH2 binding domain (MSH2bd) deleted in SLX4 ΔMSH2bd . Alignments were performed with ProViz . ( E ) HeLa FITo cells were transfected with FHA-SLX4 WT, FHA-SLX4 ΔMSH2bd or FHA-SLX4 FLW* , which is deficient for XPF binding, before FLAG IP and western blotting with the indicated antibodies.

Article Snippet: Primary antibodies against SLX4 (A302-270A, A302-269A), EXO1 (A302-640A), MSH6 (A300-022A), MSH3 (A305-314A), pS4-S8 RPA32 (A300-245A) and SMC3 (A300-060A) were purchased from Bethyl laboratories.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Control, Fractionation, Binding Assay

Journal: Nucleic Acids Research

Article Title: SLX4 dampens MutSα-dependent mismatch repair

doi: 10.1093/nar/gkac075

Figure Lengend Snippet: Characterization of the KO30 cell line expressing an N-terminally truncated form of SLX4. ( A ) Western blot showing that HeLa FITo KO1 and KO30 clones generated by CRISPR-Cas9 express a truncated form of SLX4 termed SLX4ΔNter (indicated by an arrow), the expression of which is sensitive to a siRNA that targets SLX4 mRNA. ( B ) SLX4ΔNter protein starts at Methionine 360; see supplementary results for details. ( C ) Clonogenic survival assay in response to mitomycin C (MMC) of HeLa FITo, KO30 cells and KO30 cells complemented with FHA-SLX4 WT or UBZ-mutated (UBZmut). Cells were treated for 24 h with the indicated dose of MMC ( n = 2–4 experiments, mean ± SD are represented).

Article Snippet: Primary antibodies against SLX4 (A302-270A, A302-269A), EXO1 (A302-640A), MSH6 (A300-022A), MSH3 (A305-314A), pS4-S8 RPA32 (A300-245A) and SMC3 (A300-060A) were purchased from Bethyl laboratories.

Techniques: Expressing, Western Blot, Clone Assay, Generated, CRISPR, Clonogenic Cell Survival Assay

Journal: Nucleic Acids Research

Article Title: SLX4 dampens MutSα-dependent mismatch repair

doi: 10.1093/nar/gkac075

Figure Lengend Snippet: Interaction of MSH2 and SLX4 is not required for ICL repair. ( A ) Clonogenic survival assay in response to MMC of HeLa FITo cells transfected with control siRNA (siLUC) or siRNA targeting MSH2 (siMSH2) ( n = 3 for MMC 2 ng/ml, n = 4 for MMC 5 ng/ml, mean ± SEM are represented on the graph). ( B ) Complementation of KO30 cells with FHA-SLX4 WT or SLX4 ΔMSH2bd . Induction of exogenous SLX4 expression was achieved with 2 ng/ml of doxycycline as in (C) and (D). ( C ) Clonogenic survival assay of HeLa FITo, KO30 cells and KO30 cells complemented with FHA-SLX4 WT or SLX4 ΔMSH2bd in response to MMC ( n = 3 for MMC 1 ng/ml, n = 5 or 6 for MMC 2 ng/ml, mean ± SD are represented). ( D ) Same as in (C) except that Melphalan (500 nM) was used as an alternative crosslinking agent ( n = 3, mean ± SD are indicated).

Article Snippet: Primary antibodies against SLX4 (A302-270A, A302-269A), EXO1 (A302-640A), MSH6 (A300-022A), MSH3 (A305-314A), pS4-S8 RPA32 (A300-245A) and SMC3 (A300-060A) were purchased from Bethyl laboratories.

Techniques: Clonogenic Cell Survival Assay, Transfection, Control, Expressing

Journal: Nucleic Acids Research

Article Title: SLX4 dampens MutSα-dependent mismatch repair

doi: 10.1093/nar/gkac075

Figure Lengend Snippet: Interaction of SLX4 and MSH2 contributes to the toxicity of 6-thioguanine (6-TG) or N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). ( A ) Clonogenic survival assay of HeLa FITo cells transfected with the indicated siRNA in response to a 24 h treatment with 6-TG ( n = 4–7 experiments, mean ± SEM are represented). ( B ) Clonogenic survival assay in response to 6-TG of HeLa FITo, KO30 cells and KO30 cells complemented with FHA-SLX4 WT or SLX4 ΔMSH2bd ( n = 3–5 experiments, mean ± SEM are represented). ( C ) Clonogenic survival assay in response to MNNG of KO30 cells complemented with FHA-SLX4 WT or SLX4 ΔMSH2bd ( n = 3–4 experiments, mean ± SEM are represented). ( D ) KO30 cells complemented with FHA-SLX4 WT or SLX4 ΔMSH2bd were treated with MNNG (0.1 μM) for 24 h before drug removal and addition of fresh medium. Cells were collected at the indicated time points of recovery (+rec) and induction of the DNA damage response was analysed by western blot.

Article Snippet: Primary antibodies against SLX4 (A302-270A, A302-269A), EXO1 (A302-640A), MSH6 (A300-022A), MSH3 (A305-314A), pS4-S8 RPA32 (A300-245A) and SMC3 (A300-060A) were purchased from Bethyl laboratories.

Techniques: Clonogenic Cell Survival Assay, Transfection, Western Blot

Journal: Nucleic Acids Research

Article Title: SLX4 dampens MutSα-dependent mismatch repair

doi: 10.1093/nar/gkac075

Figure Lengend Snippet: The MSH2 binding domain of SLX4 is a SHIP box that inhibits mismatch repair and antagonizes EXO1–MSH2 interaction. ( A ) Weblogo representation of multiple sequence alignments of SHIP boxes of Exo1 and Fun30 from fungal species in the Saccharomycotina ( , ). ( B ) HeLa FITo cells were transfected with expression vectors coding for GFP, FLAG-SLX4, GFP-MSH2 WT and/or GFP-MSH2 M453I , as indicated, before GFP pull-down and western blotting with the indicated antibodies. The asterisk represents the remaining signal for EXO1 when blotting for MSH3 after prior blotting for EXO1. ( C ) Peptide pull-down using biotinylated SLX4 peptides that contain a WT or mutated SHIP box, immobilized on Streptavidin-coated beads and incubated with HeLa nuclear extracts. Residues selected for mutagenesis are shown in bold in the WT sequence. ( D ) In vitro mismatch repair assay using a plasmid containing a G/T mismatch and a 5′ nick incubated with nuclear extracts (NE) from HeLa FITo cells as described in Material and Methods. Where indicated, WT or mutant SHIP peptides (80.5 μM) were added to the reaction. As a negative control, the NE was inactivated at 95°C before the reaction. DNA was purified and digested with ApalI and PvuII. Repair of the mismatch restores the PvuII site and produces two bands of 1.55 and 1.03 kb on an agarose gel. The percentage of repair is indicated.

Article Snippet: Primary antibodies against SLX4 (A302-270A, A302-269A), EXO1 (A302-640A), MSH6 (A300-022A), MSH3 (A305-314A), pS4-S8 RPA32 (A300-245A) and SMC3 (A300-060A) were purchased from Bethyl laboratories.

Techniques: Binding Assay, Sequencing, Transfection, Expressing, Western Blot, Incubation, Mutagenesis, In Vitro, Plasmid Preparation, Negative Control, Purification, Agarose Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: SLX4IP acts with SLX4 and XPF–ERCC1 to promote interstrand crosslink repair

doi: 10.1093/nar/gkz769

Figure Lengend Snippet: SLX4IP is involved in ICL repair. ( A ) Results of clonogenic survival assays conducted with HEK293A-WT and SLX4IP-KO cells exposed to DNA damage-inducing agents (mitomycin C [MMC], campothecin [CPT], ultraviolet [UV] radiation, or ionizing radiation [IR]). For each cell line, the viability of untreated cells was defined as 100%. Data are presented as the mean ± standard error of the mean (SEM; n = 3). ( B and C ) HEK293A-WT, SLX4IP-KO, XPF-KO and MUS81-KO cells were treated with the indicated concentrations of MMC for 24 h or were nontreated (NT). Cells were collected and fixed with 70% ethanol before fluorescence-activated cell sorting (FACS) analysis of the cell-cycle distribution under different treatment conditions. The mean percentages of cells in the G2/M phase from three independent repeats of FACS are shown. The results were compared statistically with those for untreated cells. Statistical analysis was performed using the Student's t -test. ** P < 0.01, *** P < 0.001, ns: not significant. P values <0.05 were considered statistically significant. ( D ) Immunostainings of SLX4, SLX4IP and γH2AX proteins performed following UV laser-induced DNA damage with the indicated antibodies. KO, knockout; WT, wild type.

Article Snippet: In this study, antibodies against the following proteins were used for western blotting and/or immunostaining: SLX4 (A302-270A; Bethyl Laboratories, Montgomery, TX, USA) or an antibody developed in-house against the antigen comprising SLX4 231-460 , XPF (A301-315A; Bethyl Laboratories), MUS81 (sc-53382; Santa Cruz Biotechnology, Dallas, TX, USA), SLX1 (ab182501; Abcam, Cambridge, UK), MSH2 (2017S; Cell Signaling Technology, Danvers, MA), SLX4IP (an antibody developed in-house against the antigen comprising SLX4IP 205-408 ), γH2AX (2577S; Cell Signaling Technology), Flag M2 (F3165-5MG; Sigma-Aldrich, St. Louis, MO, USA), α-tubulin (T6199-200UL; Sigma-Aldrich), vinculin (V9131; Sigma-Aldrich), MBP (sc-13564; Santa Cruz Biotechnology), GST (sc-138; Santa Cruz Biotechnology), ERCC1 (sc-17809; Santa Cruz Biotechnology), phospho-Chk2 (Thr68, 2661S; Cell Signaling Technology), Chk2 (2662S; Cell Signaling Technology), phospho-Chk1 (Ser345, 2348S; Cell Signaling Technology), and Chk1 (2360S; Cell Signaling Technology).

Techniques: Fluorescence, FACS, Knock-Out

Journal: Nucleic Acids Research

Article Title: SLX4IP acts with SLX4 and XPF–ERCC1 to promote interstrand crosslink repair

doi: 10.1093/nar/gkz769

Figure Lengend Snippet: SLX4IP functions in the same pathway as XPF but not as MUS81. ( A–C ) Proliferation of HEK293A-WT, SLX4IP-KO, XPF-KO, and other cell lines as indicated was measured using a CellTiter-Glo assay after 3 days in the presence of the indicated concentrations of mitomycin C (MMC). Data are presented as mean ± the standard error of the mean ( n = 3). ( D ) Experiments were done as presented in A, B, and C. Proliferation of the indicated cell lines was measured using a CellTiter-Glo assay after 3 days in the presence of the indicated concentrations of camptothecin (CPT). DKO, double knockout; IC 50 , half-maximal inhibitory concentration; KO, knockout; WT, wild type.

Article Snippet: In this study, antibodies against the following proteins were used for western blotting and/or immunostaining: SLX4 (A302-270A; Bethyl Laboratories, Montgomery, TX, USA) or an antibody developed in-house against the antigen comprising SLX4 231-460 , XPF (A301-315A; Bethyl Laboratories), MUS81 (sc-53382; Santa Cruz Biotechnology, Dallas, TX, USA), SLX1 (ab182501; Abcam, Cambridge, UK), MSH2 (2017S; Cell Signaling Technology, Danvers, MA), SLX4IP (an antibody developed in-house against the antigen comprising SLX4IP 205-408 ), γH2AX (2577S; Cell Signaling Technology), Flag M2 (F3165-5MG; Sigma-Aldrich, St. Louis, MO, USA), α-tubulin (T6199-200UL; Sigma-Aldrich), vinculin (V9131; Sigma-Aldrich), MBP (sc-13564; Santa Cruz Biotechnology), GST (sc-138; Santa Cruz Biotechnology), ERCC1 (sc-17809; Santa Cruz Biotechnology), phospho-Chk2 (Thr68, 2661S; Cell Signaling Technology), Chk2 (2662S; Cell Signaling Technology), phospho-Chk1 (Ser345, 2348S; Cell Signaling Technology), and Chk1 (2360S; Cell Signaling Technology).

Techniques: Glo Assay, Double Knockout, Concentration Assay, Knock-Out

Journal: Nucleic Acids Research

Article Title: SLX4IP acts with SLX4 and XPF–ERCC1 to promote interstrand crosslink repair

doi: 10.1093/nar/gkz769

Figure Lengend Snippet: Loss of SLX4IP reduces the repair efficiency of interstrand crosslink repair in vivo. (A) Working model of SLX4–XPF–ERCC1-SLX4IP interaction and function. Our current working hypothesis is that SLX4IP acts to promote the interaction between SLX4 and XPF–ERCC1, especially following DNA damage. This function of SLX4IP is critical for interstrand crosslink (ICL) repair. In our XPF-KO cells, truncated XPF was expressed only at low levels and SLX4IP protein was mostly degraded. Therefore, a very limited SLX4–XPF–ERCC1-SLX4IP complex formed and we observed severe ICL repair defects. When we overexpressed SLX4IP in these cells, the interaction between truncated XPF and SLX4 was enhanced, which suppressed the hypersensitivity of XPF-KO or XPF/SLX4IP-DKO cells to mitomycin C (MMC). We found that mAID-mediated downregulation of SLX4 also caused downregulation of SLX4IP protein, which may result in the failure of XPF–ERCC1 recruitment and diminished cell survival. As for SLX4IP-KO cells, SLX4 could still interact with XPF–ERCC1, but the complex was not stable; this was especially true following DNA damage, which resulted in reduced ICL repair efficiency. ( B ) Cells were treated with 1 μm MMC for 1 h and released for the indicated times. Samples were taken to detect the unhooking of ICL using a modified comet assay (please see the Material and Methods section for detailed description). DNA ICL is expressed as the percentage of decrease in Olive tail movement. Data are presented as the mean ± the standard error of the mean ( n = 3). ( C ) HEK293A cells were mock-treated (Mock) or treated with MMC (either 1# 0.5 μg/ml MMC for 1 h and release for 24 h, or 2# 0.1 μg/ml MMC for 24 h). After treatment, whole-cell extracts were prepared and subjected to Western blotting with the indicated antibodies. ( D ) HEK293A-WT, SLX4IP-KO, XPF-KO, and MUS81-KO cells were exposed to 0.5 μg/ml MMC for 1 h and allowed to recover after removal of the drug. Cells were then fixed with 3% paraformaldehyde and stained with an anti-γH2AX antibody. The percentages of cells with more than five γH2AX foci are shown. Data are presented as the mean ± the standard error of the mean ( n = 3). Statistical analysis was performed using the Student's t -test. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant. P values less than 0.05 were considered statistically significant. KO, knockout; WT, wild type.

Article Snippet: In this study, antibodies against the following proteins were used for western blotting and/or immunostaining: SLX4 (A302-270A; Bethyl Laboratories, Montgomery, TX, USA) or an antibody developed in-house against the antigen comprising SLX4 231-460 , XPF (A301-315A; Bethyl Laboratories), MUS81 (sc-53382; Santa Cruz Biotechnology, Dallas, TX, USA), SLX1 (ab182501; Abcam, Cambridge, UK), MSH2 (2017S; Cell Signaling Technology, Danvers, MA), SLX4IP (an antibody developed in-house against the antigen comprising SLX4IP 205-408 ), γH2AX (2577S; Cell Signaling Technology), Flag M2 (F3165-5MG; Sigma-Aldrich, St. Louis, MO, USA), α-tubulin (T6199-200UL; Sigma-Aldrich), vinculin (V9131; Sigma-Aldrich), MBP (sc-13564; Santa Cruz Biotechnology), GST (sc-138; Santa Cruz Biotechnology), ERCC1 (sc-17809; Santa Cruz Biotechnology), phospho-Chk2 (Thr68, 2661S; Cell Signaling Technology), Chk2 (2662S; Cell Signaling Technology), phospho-Chk1 (Ser345, 2348S; Cell Signaling Technology), and Chk1 (2360S; Cell Signaling Technology).

Techniques: In Vivo, Modification, Single Cell Gel Electrophoresis, Western Blot, Staining, Knock-Out

Journal: Nucleic Acids Research

Article Title: SLX4IP acts with SLX4 and XPF–ERCC1 to promote interstrand crosslink repair

doi: 10.1093/nar/gkz769

Figure Lengend Snippet: SLX4IP stabilizes the interaction between SLX4 and XPF–ERCC1, especially after DNA damage. ( A ) HEK293A-WT and SLX4IP-KO cells were nontreated (NT) or treated with 1 μm mitomycin C (MMC) for 24 h, and radiated with 100 J/m 2 ultraviolet (UV) radiation and released for 4 h. Whole-cell extracts were analyzed by gel filtration on a Superdex 200 10/300 GL preparative-grade column. 96 fractions were collected in volumes of 200 μl. Fractions as indicated were analyzed with antibodies as indicated. ( B ) SLX4 was subjected to immunoprecipitation (IP) with anti-SLX4 serum using lysates of HEK293A-WT cells, XPF-KO cells, and other indicated cell lines. ( C ) The same amount of GFP or GFP-SLX4 293-638 was mixed with GFP beads. After rotation in a cold room for 1 h, protein-bound beads were washed with NETN lysis buffer. The indicated amounts of XPF and/or SLX4IP were mixed with NETN lysis buffer to the same volume, and the mixture was allowed to bind to protein bound on GFP beads. After mixing in a cold room for 1 h, beads were thoroughly washed and boiled at 95°C in Laemmli buffer. GFP and GFP-SLX4 293-638 were detected using Coomassie Blue staining. XPF was detected using an anti-GST antibody, and SLX4IP was detected using an anti-MBP antibody. Quantification analysis was done with ImageJ and the amount of relative XPF protein bound to the beads is shown. DKO, double knockout; KO, knockout; WT, wild type.

Article Snippet: In this study, antibodies against the following proteins were used for western blotting and/or immunostaining: SLX4 (A302-270A; Bethyl Laboratories, Montgomery, TX, USA) or an antibody developed in-house against the antigen comprising SLX4 231-460 , XPF (A301-315A; Bethyl Laboratories), MUS81 (sc-53382; Santa Cruz Biotechnology, Dallas, TX, USA), SLX1 (ab182501; Abcam, Cambridge, UK), MSH2 (2017S; Cell Signaling Technology, Danvers, MA), SLX4IP (an antibody developed in-house against the antigen comprising SLX4IP 205-408 ), γH2AX (2577S; Cell Signaling Technology), Flag M2 (F3165-5MG; Sigma-Aldrich, St. Louis, MO, USA), α-tubulin (T6199-200UL; Sigma-Aldrich), vinculin (V9131; Sigma-Aldrich), MBP (sc-13564; Santa Cruz Biotechnology), GST (sc-138; Santa Cruz Biotechnology), ERCC1 (sc-17809; Santa Cruz Biotechnology), phospho-Chk2 (Thr68, 2661S; Cell Signaling Technology), Chk2 (2662S; Cell Signaling Technology), phospho-Chk1 (Ser345, 2348S; Cell Signaling Technology), and Chk1 (2360S; Cell Signaling Technology).

Techniques: Filtration, Immunoprecipitation, Lysis, Staining, Double Knockout, Knock-Out

Journal: bioRxiv

Article Title: RNA transcripts suppress G-quadruplex structures through G-loop formation

doi: 10.1101/2023.03.09.531892

Figure Lengend Snippet: a , pG4 BOT and pPolyT were incubated in NPE pre-treated with buffer or RNaseA, and products were analysed by ChIP-qPCR with the FANCD2 antibody and a primer pair for the G4 locus. The relative values compared to the highest signal among the conditions were plotted. b , Mock- and FANCA-depleted NPEs were analysed by Western blot with the FANCA antibody. The asterisk represents a non-specific band. c , pG4 BOT was incubated in the NPEs as described in b , and products were analysed by ChIP-qPCR as in a with XPF (i) and SLX4 (ii) antibodies using primers for the G4 locus (schematic, right). d , Mock-, XPF-, MUS81-, SLX1-, and XPA-depleted NPEs supplemented with buffer, or where indicated with the XPF-ERCC1 complex or SLX4, were analysed by Western blot with XPF, MUS81, SLX1, SLX4, and XPA antibodies (bottom). The asterisks represent non-specific bands. Schematic representation of SLX4 and its interacting nucleases is depicted (top). e , pG4 TOP was incubated in the NPEs as described in d , and products were digested with AflIII, end-labelled, separated by denaturing PAGE alongside a sequencing ladder, and visualized by autoradiography. Incised fragments (−1 to −8) are indicated with a bracket. f , Mock- and DHX36-FANCJ-depleted NPEs supplemented with buffer or wild-type DHX36 and FANCJ were analysed by Western blot with DHX36 and FANCJ antibodies. g, pG4 TOP was incubated in the NPEs as described in f, and products were analysed by denaturing PAGE as in e . h, pG4 BOT was incubated in the NPEs as described in f , and products were analysed by DRIP- qPCR using primers for the G4 locus. Relative values compared to input signals were plotted. i, pPolyT and pG4 BOT were incubated in NPE, and products were analysed by ChIP-qPCR with DHX36 (i) and FANCJ (ii) antibodies using primers for the G4 locus (schematic, right). Where indicated, NPE was pre-treated with RNase A. The relative values compared to the highest signal among the conditions were plotted.

Article Snippet: Antibodies against xl BRCA2 , xl DHX36 , xl FANCD2 , xl FANCJ , xl MUS81 , hs RAD51 , xl SLX1 , xl SLX4 , xl XPA , xl XPF and hs RNA Polymerase II subunit A (Bethyl) were previously described.

Techniques: Incubation, ChIP-qPCR, Western Blot, Sequencing, Autoradiography

Journal: Nucleic Acids Research

Article Title: SLX4IP acts with SLX4 and XPF–ERCC1 to promote interstrand crosslink repair

doi: 10.1093/nar/gkz769

Figure Lengend Snippet: The conserved N-terminus of SLX4IP is responsible for its interaction with SLX4–XPF–ERCC1. ( A ) HEK293A cells were transfected with constructs encoding SLX4IP-SFB or its deletion variants and subjected to immunoprecipitation (IP) pulldown with S beads. Western blotting conducted with antibodies as indicated. A schematic overview of the deletion strategy and protein-protein interaction results is presented. ( B ) Conservation of sequences at the N-terminus of SLX4IP. The alignment of the N-terminal sequences of SLX4IP in humans, mice, zebrafish and Xenopus , established using the Clustal Omega sequence alignment program, is shown. ( C ) HEK293A cells were transfected with constructs encoding SLX4IP-SFB or its point mutation variants and subjected to IP with S beads. Western blotting was conducted with antibodies as indicated. ( D ) SLX4 was subjected to IP with anti-SLX4 serum from HEK293A-WT or SLX4IP-KO cells (clone #6 and clone #13). Western blotting was conducted with antibodies as indicated. ( E ) HEK293A-WT or SLX4IP-KO cells were transfected with SFB-XPF and subjected to IP with S beads. Western blotting was conducted with antibodies as indicated. ( F ) Western blots detecting the interaction between XPF and SLX4 in HEK293A-WT and SLX4IP-KO cells were analyzed by ImageJ. The mean relative interaction from five experiments is shown. FL, full length (controls); KO, knockout; WT, wild type.

Article Snippet: In this study, antibodies against the following proteins were used for western blotting and/or immunostaining: SLX4 (A302-270A; Bethyl Laboratories, Montgomery, TX, USA) or an antibody developed in-house against the antigen comprising SLX4 231-460 , XPF (A301-315A; Bethyl Laboratories), MUS81 (sc-53382; Santa Cruz Biotechnology, Dallas, TX, USA), SLX1 (ab182501; Abcam, Cambridge, UK), MSH2 (2017S; Cell Signaling Technology, Danvers, MA), SLX4IP (an antibody developed in-house against the antigen comprising SLX4IP 205-408 ), γH2AX (2577S; Cell Signaling Technology), Flag M2 (F3165-5MG; Sigma-Aldrich, St. Louis, MO, USA), α-tubulin (T6199-200UL; Sigma-Aldrich), vinculin (V9131; Sigma-Aldrich), MBP (sc-13564; Santa Cruz Biotechnology), GST (sc-138; Santa Cruz Biotechnology), ERCC1 (sc-17809; Santa Cruz Biotechnology), phospho-Chk2 (Thr68, 2661S; Cell Signaling Technology), Chk2 (2662S; Cell Signaling Technology), phospho-Chk1 (Ser345, 2348S; Cell Signaling Technology), and Chk1 (2360S; Cell Signaling Technology).

Techniques: Transfection, Construct, Immunoprecipitation, Western Blot, Sequencing, Mutagenesis, Knock-Out

Journal: Nucleic Acids Research

Article Title: SLX4IP acts with SLX4 and XPF–ERCC1 to promote interstrand crosslink repair

doi: 10.1093/nar/gkz769

Figure Lengend Snippet: The helicase-like domain of XPF is responsible for its interaction with SLX4 and SLX4IP. ( A ) HEK293A-WT or XPF-KO cells were transfected with SLX4IP-SFB and subjected to immunoprecipitation (IP) with S beads. Western blotting was conducted with antibodies as indicated. ( B ) A schematic overview of the XPF domains, XPF deletion mutants, and results of protein-protein interactions is presented. ( C and D ) XPF-KO cells were transfected with a construct encoding SFB-XPF, a deletion mutant of SFB-XPF, or a point mutation variant of SFB-XPF along with a construct encoding GFP-SLX4 ( C ) or GFP-SLX4IP ( D ). Cell lysates were subjected to IP with S beads. Western blotting was conducted with antibodies as indicated. ( E ) A schematic model shows the interaction domain of SLX4–XPF–ERCC1-SLX4IP. ( F ) To induce degradation of SLX4-mAID-SFB protein, 1 mM auxin (IAA) was added to cells for 48 h. Western blotting was conducted with antibodies as indicated. ( G ) HEK293A SLX4-mAID-SFB knock-in (KI) cells were infected with a virus encoding HA-SLX4IP and treated with or without auxin for 48 h. Cells were collected, lysed, and subjected to IP with hemagglutinin (HA) beads. Western blotting was conducted with antibodies as indicated. ( H ) HEK293A cells were infected with a virus encoding inducible control shRNA or shRNA for SLX4 (#84 and #85). To induce knockdown of SLX4 protein, 1 μg/ml of doxycycline (DOX) was added to cells for 48 h. Western blotting was conducted to detect the levels of the indicated proteins in whole-cell extract and chromatin extracts. ( I ) The expression levels of proteins in HEK293A-WT, SLX4IP-KO, XPF-KO and XPF/SLX4IP-DKO cells were detected with antibodies as indicated. HLD, helicase-like domain; KO, knockout; WT, wild type.

Article Snippet: In this study, antibodies against the following proteins were used for western blotting and/or immunostaining: SLX4 (A302-270A; Bethyl Laboratories, Montgomery, TX, USA) or an antibody developed in-house against the antigen comprising SLX4 231-460 , XPF (A301-315A; Bethyl Laboratories), MUS81 (sc-53382; Santa Cruz Biotechnology, Dallas, TX, USA), SLX1 (ab182501; Abcam, Cambridge, UK), MSH2 (2017S; Cell Signaling Technology, Danvers, MA), SLX4IP (an antibody developed in-house against the antigen comprising SLX4IP 205-408 ), γH2AX (2577S; Cell Signaling Technology), Flag M2 (F3165-5MG; Sigma-Aldrich, St. Louis, MO, USA), α-tubulin (T6199-200UL; Sigma-Aldrich), vinculin (V9131; Sigma-Aldrich), MBP (sc-13564; Santa Cruz Biotechnology), GST (sc-138; Santa Cruz Biotechnology), ERCC1 (sc-17809; Santa Cruz Biotechnology), phospho-Chk2 (Thr68, 2661S; Cell Signaling Technology), Chk2 (2662S; Cell Signaling Technology), phospho-Chk1 (Ser345, 2348S; Cell Signaling Technology), and Chk1 (2360S; Cell Signaling Technology).

Techniques: Transfection, Immunoprecipitation, Western Blot, Protein-Protein interactions, Construct, Mutagenesis, Variant Assay, Knock-In, Infection, Virus, Control, shRNA, Knockdown, Expressing, Knock-Out

Journal: Nucleic Acids Research

Article Title: SLX4IP acts with SLX4 and XPF–ERCC1 to promote interstrand crosslink repair

doi: 10.1093/nar/gkz769

Figure Lengend Snippet: SLX4IP stabilizes the interaction between SLX4 and XPF–ERCC1, especially after DNA damage. ( A ) HEK293A-WT and SLX4IP-KO cells were nontreated (NT) or treated with 1 μm mitomycin C (MMC) for 24 h, and radiated with 100 J/m 2 ultraviolet (UV) radiation and released for 4 h. Whole-cell extracts were analyzed by gel filtration on a Superdex 200 10/300 GL preparative-grade column. 96 fractions were collected in volumes of 200 μl. Fractions as indicated were analyzed with antibodies as indicated. ( B ) SLX4 was subjected to immunoprecipitation (IP) with anti-SLX4 serum using lysates of HEK293A-WT cells, XPF-KO cells, and other indicated cell lines. ( C ) The same amount of GFP or GFP-SLX4 293-638 was mixed with GFP beads. After rotation in a cold room for 1 h, protein-bound beads were washed with NETN lysis buffer. The indicated amounts of XPF and/or SLX4IP were mixed with NETN lysis buffer to the same volume, and the mixture was allowed to bind to protein bound on GFP beads. After mixing in a cold room for 1 h, beads were thoroughly washed and boiled at 95°C in Laemmli buffer. GFP and GFP-SLX4 293-638 were detected using Coomassie Blue staining. XPF was detected using an anti-GST antibody, and SLX4IP was detected using an anti-MBP antibody. Quantification analysis was done with ImageJ and the amount of relative XPF protein bound to the beads is shown. DKO, double knockout; KO, knockout; WT, wild type.

Article Snippet: In this study, antibodies against the following proteins were used for western blotting and/or immunostaining: SLX4 (A302-270A; Bethyl Laboratories, Montgomery, TX, USA) or an antibody developed in-house against the antigen comprising SLX4 231-460 , XPF (A301-315A; Bethyl Laboratories), MUS81 (sc-53382; Santa Cruz Biotechnology, Dallas, TX, USA), SLX1 (ab182501; Abcam, Cambridge, UK), MSH2 (2017S; Cell Signaling Technology, Danvers, MA), SLX4IP (an antibody developed in-house against the antigen comprising SLX4IP 205-408 ), γH2AX (2577S; Cell Signaling Technology), Flag M2 (F3165-5MG; Sigma-Aldrich, St. Louis, MO, USA), α-tubulin (T6199-200UL; Sigma-Aldrich), vinculin (V9131; Sigma-Aldrich), MBP (sc-13564; Santa Cruz Biotechnology), GST (sc-138; Santa Cruz Biotechnology), ERCC1 (sc-17809; Santa Cruz Biotechnology), phospho-Chk2 (Thr68, 2661S; Cell Signaling Technology), Chk2 (2662S; Cell Signaling Technology), phospho-Chk1 (Ser345, 2348S; Cell Signaling Technology), and Chk1 (2360S; Cell Signaling Technology).

Techniques: Filtration, Immunoprecipitation, Lysis, Staining, Double Knockout, Knock-Out

Journal: Nucleic Acids Research

Article Title: SLX4IP acts with SLX4 and XPF–ERCC1 to promote interstrand crosslink repair

doi: 10.1093/nar/gkz769

Figure Lengend Snippet: Loss of SLX4IP reduces the repair efficiency of interstrand crosslink repair in vivo. (A) Working model of SLX4–XPF–ERCC1-SLX4IP interaction and function. Our current working hypothesis is that SLX4IP acts to promote the interaction between SLX4 and XPF–ERCC1, especially following DNA damage. This function of SLX4IP is critical for interstrand crosslink (ICL) repair. In our XPF-KO cells, truncated XPF was expressed only at low levels and SLX4IP protein was mostly degraded. Therefore, a very limited SLX4–XPF–ERCC1-SLX4IP complex formed and we observed severe ICL repair defects. When we overexpressed SLX4IP in these cells, the interaction between truncated XPF and SLX4 was enhanced, which suppressed the hypersensitivity of XPF-KO or XPF/SLX4IP-DKO cells to mitomycin C (MMC). We found that mAID-mediated downregulation of SLX4 also caused downregulation of SLX4IP protein, which may result in the failure of XPF–ERCC1 recruitment and diminished cell survival. As for SLX4IP-KO cells, SLX4 could still interact with XPF–ERCC1, but the complex was not stable; this was especially true following DNA damage, which resulted in reduced ICL repair efficiency. ( B ) Cells were treated with 1 μm MMC for 1 h and released for the indicated times. Samples were taken to detect the unhooking of ICL using a modified comet assay (please see the Material and Methods section for detailed description). DNA ICL is expressed as the percentage of decrease in Olive tail movement. Data are presented as the mean ± the standard error of the mean ( n = 3). ( C ) HEK293A cells were mock-treated (Mock) or treated with MMC (either 1# 0.5 μg/ml MMC for 1 h and release for 24 h, or 2# 0.1 μg/ml MMC for 24 h). After treatment, whole-cell extracts were prepared and subjected to Western blotting with the indicated antibodies. ( D ) HEK293A-WT, SLX4IP-KO, XPF-KO, and MUS81-KO cells were exposed to 0.5 μg/ml MMC for 1 h and allowed to recover after removal of the drug. Cells were then fixed with 3% paraformaldehyde and stained with an anti-γH2AX antibody. The percentages of cells with more than five γH2AX foci are shown. Data are presented as the mean ± the standard error of the mean ( n = 3). Statistical analysis was performed using the Student's t -test. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant. P values less than 0.05 were considered statistically significant. KO, knockout; WT, wild type.

Article Snippet: In this study, antibodies against the following proteins were used for western blotting and/or immunostaining: SLX4 (A302-270A; Bethyl Laboratories, Montgomery, TX, USA) or an antibody developed in-house against the antigen comprising SLX4 231-460 , XPF (A301-315A; Bethyl Laboratories), MUS81 (sc-53382; Santa Cruz Biotechnology, Dallas, TX, USA), SLX1 (ab182501; Abcam, Cambridge, UK), MSH2 (2017S; Cell Signaling Technology, Danvers, MA), SLX4IP (an antibody developed in-house against the antigen comprising SLX4IP 205-408 ), γH2AX (2577S; Cell Signaling Technology), Flag M2 (F3165-5MG; Sigma-Aldrich, St. Louis, MO, USA), α-tubulin (T6199-200UL; Sigma-Aldrich), vinculin (V9131; Sigma-Aldrich), MBP (sc-13564; Santa Cruz Biotechnology), GST (sc-138; Santa Cruz Biotechnology), ERCC1 (sc-17809; Santa Cruz Biotechnology), phospho-Chk2 (Thr68, 2661S; Cell Signaling Technology), Chk2 (2662S; Cell Signaling Technology), phospho-Chk1 (Ser345, 2348S; Cell Signaling Technology), and Chk1 (2360S; Cell Signaling Technology).

Techniques: In Vivo, Modification, Single Cell Gel Electrophoresis, Western Blot, Staining, Knock-Out

Journal: Communications Biology

Article Title: TopBP1 coordinates DNA repair synthesis in mitosis via recruitment of the nuclease scaffold SLX4

doi: 10.1038/s42003-025-08442-9

Figure Lengend Snippet: A , B Representative images of native metaphase spreads showing the colocalization of TopBP1 and EdU with FANCD2 or SLX4 on CAPH-marked chromatin after mild replication stress (0.4 µM APH, 16 h) in HeLa Kyoto cell line (NCAPH-mEGFP, TopBP1-Halo). CDK1i (RO-3306) was used for G2-synchronization prior to mitotic release. White arrows indicate foci on sister chromatids in boxed zoom. C Western blot showing degradation of endogenous TopBP1 after activation of mAID-SMASh double-degron tag in RPE1 cells. D Representative images and quantification of TopBP1, EdU, and FANCD2 foci in prometaphase cells. Statistical significance was evaluated by Student’s t -test ( n = 66 (control) or 100 (+IAA)). Horizontal lines in violin plots indicate median and dotted lines indicate quartiles. CDK1i (RO-3306) was used for G2-synchronization prior to mitotic release. E Quantification of FANCD2 foci in the experiment described in panel D .

Article Snippet: After blocking, the membrane was incubated with primary antibodies against TopBP1 (1:2000, A300-111A, Bethyl Laboratories), SLX4 (1:1000, gift from John Rouse, University of Dundee), and Tubulin (1:2000, Abcam, ab6160) for 1 h at room temperature.

Techniques: Western Blot, Activation Assay, Control

Journal: Communications Biology

Article Title: TopBP1 coordinates DNA repair synthesis in mitosis via recruitment of the nuclease scaffold SLX4

doi: 10.1038/s42003-025-08442-9

Figure Lengend Snippet: A Representative images and quantification of TopBP1 twin foci per prometaphase (chromosomes condensed but not yet aligned along the metaphase plate) cell after untreated or replication stress conditions in HeLa Kyoto cell line (NCAPH-mEGFP, TopBP1-Halo). Orange arrows indicate TopBP1 twin foci. Statistical significance was evaluated by Student’s t -test ( n = 48 (control) or 52 (APH)). Horizontal lines in violin plots indicate median and dotted lines indicate quartiles. Notably, CDK1-inhibition was not used in this experiment. B Representative images of TopBP1 and FANCD2 twin foci in early mitosis (prophase/prometaphase). Dashed squares indicate two zoom regions containing colocalizing twin foci of TopBP1 and FANCD2 on condensed DNA (DAPI). Quantification of relative TopBP1 and FANCD2 focus intensities are shown in bar plot. A indicates the sister chromatid containing the most intense TopBP1 focus, while B indicates the sister chromatid with the less intense TopBP1 focus. Each data point represents quantification from one focus ( n = 37, 2 biological replicates). Notably, CDK1-inhibition was not used in this experiment. C Representative image of a native metaphase spread showing the localization of TopBP1 and FANCD2 on chromatin (visualized by CAPH). Numbers indicate sister chromatids shown in zoom view. 1–4 indicates four different types of TopBP1 and FANCD2 localization on chromatin (between sister chromatids, on chromatin, in gaps, and on telomeres) after mild replication stress (0.4 µM APH, 16 h). White arrow indicates a gap on the chromatin. D Representative images and illustrations of TopBP1-FANCD2 localization patterns on metaphase chromatin. A1–A4 illustrate asymmetric localization patterns between TopBP1 and FANCD2. S1–S4 illustrate symmetric localization patterns between TopBP1 and FANCD2. White arrows indicate the TopBP1-FANCD2 localization pattern illustrated below images. E Quantification of TopBP1-FANCD2 symmetry from data presented in ( C ). Pie-chart shows the percentages of symmetric/asymmetric patterns of TopBP1-FANCD2 foci on metaphase chromatin. Bar-plots show the percentage contribution of the different symmetric (S1–S4) and asymmetric (A1–A4) TopBP1-FANCD2 structures ( n = 458). C – E CDK1i (RO-3306) was used for G2-synchronization prior to mitotic release.

Article Snippet: After blocking, the membrane was incubated with primary antibodies against TopBP1 (1:2000, A300-111A, Bethyl Laboratories), SLX4 (1:1000, gift from John Rouse, University of Dundee), and Tubulin (1:2000, Abcam, ab6160) for 1 h at room temperature.

Techniques: Control, Inhibition

Journal: Communications Biology

Article Title: TopBP1 coordinates DNA repair synthesis in mitosis via recruitment of the nuclease scaffold SLX4

doi: 10.1038/s42003-025-08442-9

Figure Lengend Snippet: A Illustrations of TopBP1-FANCD2-EdU localization patterns on metaphase chromatin in the HeLa Kyoto cell line (NCAPH-mEGFP, TopBP1-Halo). A1–A8 illustrate asymmetric localization patterns between TopBP1, FANCD2, and EdU. S1–S2 illustrate symmetric localization patterns between TopBP1, FANCD2, and EdU. B Quantification of TopBP1-FANCD2-EdU symmetry. Pie-chart shows the percentages of symmetric/asymmetric patterns of TopBP1-FANCD2-EdU foci on metaphase chromatin. Bar-plots show the percentage contribution of the different symmetric (S1–S2) and asymmetric (A1–A8) TopBP1-FANCD2-EdU structures ( n = 163). C Representative images of the two most highly represented symmetric/asymmetric localization patterns of TopBP1, FANCD2, and EdU. White box indicates area of zoom. White arrow indicates foci that colocalize with TopBP1. D Representative image of asymmetric structure A4. Dashed circles indicate areas of quantification, where intensities of TopBP1, FANCD2, and EdU were measured. A indicates the sister chromatid containing the most intense TopBP1 focus, while B indicates the sister chromatid lacking (or with very dim) TopBP1 focus. Quantification of relative TopBP1, FANCD2, and EdU focus intensities are shown in bar plot. Each triangle indicates quantification from an A4 asymmetric structure ( n = 15, 2 biological replicates). B – D CDK1i (RO-3306) was used for G2-synchronization prior to mitotic release.

Article Snippet: After blocking, the membrane was incubated with primary antibodies against TopBP1 (1:2000, A300-111A, Bethyl Laboratories), SLX4 (1:1000, gift from John Rouse, University of Dundee), and Tubulin (1:2000, Abcam, ab6160) for 1 h at room temperature.

Techniques:

Journal: Communications Biology

Article Title: TopBP1 coordinates DNA repair synthesis in mitosis via recruitment of the nuclease scaffold SLX4

doi: 10.1038/s42003-025-08442-9

Figure Lengend Snippet: A Experimental scheme and representative images of TopBP1 and EdU foci on CAPH-visualized metaphase chromosomes with or without high-dose APH (2 µM, 1 h) during EdU pulse in early mitosis in HeLa Kyoto cell line (NCAPH-mEGFP, TopBP1-Halo). B Quantification of total EdU foci per metaphase spread with or without high-dose APH (2 µM, 1 h). Statistical significance was evaluated by Student’s t -test. ****, p < 0.0001 ( n = 77). Horizontal lines in violin plots indicate median and dotted lines indicate quartiles. C Quantification of total TopBP1 foci per metaphase spread with or without high-dose APH (2 µM, 1 h). Statistical significance was evaluated by Student’s t -test ( n = 77). Horizontal lines in violin plots indicate median and dotted lines indicate quartiles. ns not significant. D Quantification of relative intensity of TopBP1 in twin foci under inhibition of MiDAS with or without high-dose APH (2 µM, 1 h). A indicates the sister chromatid containing the brighter TopBP1 focus, while ( B ) indicates the sister chromatid with the dimmer TopBP1 focus. Each triangle in the bar plot indicates quantification of a pair of TopBP1 twin foci from ( A ) ( n = 20 (Control) or n = 34 (2 µM APH). Two biological replicates. E Quantification of the localization of TopBP1 relative to chromatin gaps. “Colocalizing” indicates the percentage of chromatin gaps that contain a TopBP1 focus. “Non-colocalizing” indicates chromatin gaps devoid of TopBP1 focus. n = 196 indicates that 196 chromatin gaps were evaluated for colocalization with TopBP1. Error bars indicate 95% confidence intervals. F Quantification showing the percentage of TopBP1-bound chromatin gaps that are EdU positive or negative. n = 50 indicates that 50 TopBP1-bound chromosome gaps were evaluated for colocalization with EdU. G Quantification of TopBP1 positive chromatin gaps per metaphase spread with or without high-dose APH (2 µM, 1 h). Statistical significance was evaluated by Student’s t -test ( n = 23). Horizontal lines in violin plots indicate median and dotted lines indicate quartiles. ns not significant. B – G CDK1i (RO-3306) was used for G2-synchronization prior to mitotic release.

Article Snippet: After blocking, the membrane was incubated with primary antibodies against TopBP1 (1:2000, A300-111A, Bethyl Laboratories), SLX4 (1:1000, gift from John Rouse, University of Dundee), and Tubulin (1:2000, Abcam, ab6160) for 1 h at room temperature.

Techniques: Inhibition, Control

Journal: Communications Biology

Article Title: TopBP1 coordinates DNA repair synthesis in mitosis via recruitment of the nuclease scaffold SLX4

doi: 10.1038/s42003-025-08442-9

Figure Lengend Snippet: A Illustration of human TopBP1 domain organization. Blue boxes indicate BRCT-domains capable of binding phosphorylated residues through an internal lysine residue. Schematic summary of Dpb11-Slx4 interaction in yeast and hypothesis for interaction in human. SR (Slx1-Rad1). SMX (SLX1-MUS81-XPF). B Representative images of HeLa cells expressing mCherry-tagged WT or mutant (K704A) TopBP1 cDNA from FRT site, treated with APH (0.4 µM) for 16 h before imaging. Cells were subjected to knockdown of SLX4 from endogenous locus with siRNA’s targeting 5′ and 3′ UTR and complemented with Venus-tagged SLX4 cDNA by transient transfection. Notably, knockdown of endogenous SLX4 was performed twice. White arrows indicate colocalizing foci in TopBP1-K704A cells. White lines crossing TopBP1-SLX4 foci indicate the line used for quantification of TopBP1-SLX4 intensity values (gray value) plotted in ( C ). Notably, CDK1-inhibition was not used in this experiment. C Plot showing the intensity values of TopBP1 (WT or K704A) and SLX4 (WT) along the white lines indicated in ( B ). White arrows indicate the two foci that are intersected by the white line from the K704A images in ( B ). Dashed black line indicates the background intensity of SLX4. D Quantification of colocalizing focus intensities. Plotted values indicate the intensity-ratio between SLX4 and TopBP1 at colocalizing foci, normalized to the total SLX4 signal per nucleus. Horizontal lines in violin plots indicate median and dotted lines indicate quartiles ( n = 34 (WT) or n = 37 (K704A), 2 replicates).

Article Snippet: After blocking, the membrane was incubated with primary antibodies against TopBP1 (1:2000, A300-111A, Bethyl Laboratories), SLX4 (1:1000, gift from John Rouse, University of Dundee), and Tubulin (1:2000, Abcam, ab6160) for 1 h at room temperature.

Techniques: Binding Assay, Residue, Expressing, Mutagenesis, Imaging, Knockdown, Transfection, Inhibition

Journal: Communications Biology

Article Title: TopBP1 coordinates DNA repair synthesis in mitosis via recruitment of the nuclease scaffold SLX4

doi: 10.1038/s42003-025-08442-9

Figure Lengend Snippet: A Illustration of human SLX4 domain organization with motifs and important residues indicated. ∆SIM mutations are compared to WT sequence. B Representative images of mitotic HeLa cells expressing Halo-tagged TopBP1 from the endogenous locus, TFP-tagged H2B and mCherry-tagged WT or mutant SLX4 cDNA. Cells were treated with APH (0.4 µM) for 16 h before imaging. Endogenous SLX4 was depleted before APH treatment, as described in Fig. . White lines crossing TopBP1-SLX4 foci indicate the line used for quantification of TopBP1-SLX4 intensity values (gray value) plotted in ( C ). C Plots showing the intensity values of TopBP1 (WT) and SLX4 (WT, T1260A, ∆SIM1-2 or ∆SIM1-3) fluorophores along the white lines indicated in ( B ). D Quantification of the percentage of cells with colocalization of at least one SLX4 (WT or mutant) focus with the TopBP1 foci. Colored bullets indicate data from three independent experiments. Mean and error bars indicating 95% confidence-intervals are shown (Student’s t -test). E Quantification of the percentage of cells with colocalization of at least one SLX4 focus with the TopBP1 foci in cells expressing either SLX4-WT or SLX4-T1260A peptides. Colored bullets indicate data from three independent experiments with 3 different clones. Mean and error bars indicating 95% confidence-intervals are shown (Student’s t -test). Statistical test based on Student’s t -test ( n = 72 (SLX4-WT) or n = 66 (SLX4-T1260A)). *, p < 0.05. F Quantification of fluorescence intensities of colocalizing TopBP1 and SLX4 foci in cells expressing either SLX4-WT or SLX4-T1260A peptides. Plotted values indicate the intensity-ratio between TopBP1 and SLX4 at colocalizing foci, normalized to the total SLX4 signal per nucleus. Data is plotted as super-plot in three colors, indicating data points from three independent experiments with three different clones. Mean and error bars indicating 95% confidence intervals are shown. Statistical test based on Student’s t -test ( n = 327 (SLX4-WT) or n = 299 (SLX4-T1260A)). ****, p < 0.0001. G Quantification of nuclear SUMO2/3 staining intensity in control condition or after treatment with TAK-981 at indicated concentrations. Colored bullets indicate data from three independent experiments. >100 cells per condition were quantified for each replica by automated image analysis. Mean and error bars indicating 95% confidence-intervals are shown. Statistics were evaluated by Student’s t -test. ***, p < 0.001. ****, p < 0.0001. H Representative images of mitotic cells expressing Halo-tagged TopBP1 from the endogenous locus, TFP-tagged H2B and mCherry-tagged WT SLX4 cDNA in the absence or presence of TAK-981 (4 h treatment). Endogenous SLX4 was depleted prior to treatment, as described in Fig. . White lines crossing TopBP1-SLX4 foci indicate the line used for quantification of TopBP1-SLX4 intensity values (gray value) plotted in ( I ). White arrows indicate the two foci, which are intersected by the white line. I Plot showing the intensity values of TopBP1 (WT) and SLX4 (WT) along the white lines indicated in ( H ). Dashed black line indicates the background intensity of SLX4. J Quantification of fluorescence intensities of colocalizing TopBP1 and SLX4 foci in the absence or presence of TAK-981 (quantified from data in ( H )). Plotted values indicate the intensity-ratio between TopBP1 and SLX4 at colocalizing foci, normalized to the total SLX4 signal per nucleus. Data is plotted as super-plot in two colors, indicating data points from the two independent experiments. Means are indicated by horizontal lines ( n = 131 (Control) or n = 103 (TAK-981)). K Quantification of total EdU foci per metaphase spread with low dose APH (0.4 µM, 16 h) and with or without SUMO-inhibition by TAK-981 (1 µM, 2 h). Analysis performed on cells synchronized with CDK1 inhibitor RO-3306 or without RO-3306. Mean and error bars indicating 95% confidence intervals are shown. Statistical test based on Student’s t -test (RO-33306, n = 284 (DMSO) or n = 292 (TAK-981); No RO-3306, n = 63 (DMSO) or n = 63 (TAK-981)). ****, p < 0.0001. B – J CDK1-inhibition was not used in these experiments.

Article Snippet: After blocking, the membrane was incubated with primary antibodies against TopBP1 (1:2000, A300-111A, Bethyl Laboratories), SLX4 (1:1000, gift from John Rouse, University of Dundee), and Tubulin (1:2000, Abcam, ab6160) for 1 h at room temperature.

Techniques: Sequencing, Expressing, Mutagenesis, Imaging, Clone Assay, Fluorescence, Staining, Control, Inhibition

Journal: Communications Biology

Article Title: TopBP1 coordinates DNA repair synthesis in mitosis via recruitment of the nuclease scaffold SLX4

doi: 10.1038/s42003-025-08442-9

Figure Lengend Snippet: A Experimental scheme. Mitotic cells were reseeded in suspension into an 8-well chambered Ibidi slide in DMEM (10% FBS, 1% P/S), followed by 4 h incubation for the cells to adhere to the bottom. Hereafter, cells were fixed for 10 min in 4% formaldehyde and stained for 53BP1. B , C Quantification of mitotic cells with EdU foci (in percentage) in cells expressing WT or mutant variants of TopBP1 or SLX4. Expression of cDNA was induced by DOX after knockdown of endogenous TopBP1 or SLX4 using siRNA targeting the 3′ UTR or 5′ and 3′ UTR, respectively (see Western blots in Supplementary Fig. ). Expression of WT or mutant TopBP1 or SLX4 cDNA from an FRT-site were performed as outlined in ( A ) ( n = 2). > 300 cells were quantified per condition across the two replicates. CDK1i (RO-3306) was used for G2-synchronization prior to mitotic release. Means are indicated by horizontal lines. D Representative images of immunofluorescence staining for 53BP1 foci in G 1 cells after transition through mitosis with TopBP1 K704A as outlined in ( A ). DAPI was used as counterstain. E Quantification of 53BP1 nuclear bodies per G 1 cell in images from ( D ). Mean and error bars indicating 95% confidence intervals are shown. Statistical test based on Student’s t -test ( n = 287 (WT) or 284 (K704A). ****, p < 0.0001). F Representative images of immunofluorescence staining for 53BP1 foci in G 1 cells after transition through mitosis with SLX4 T1260A as outlined in ( A ). DAPI was used as counterstain. G Quantification of 53BP1 nuclear bodies per G 1 cell in images from ( F ). Mean and error bars indicating 95% confidence intervals are shown. Statistical test based on Student’s t -test ( n = 287 (WT) or 278 (T1260A). ****, p < 0.0001).

Article Snippet: After blocking, the membrane was incubated with primary antibodies against TopBP1 (1:2000, A300-111A, Bethyl Laboratories), SLX4 (1:1000, gift from John Rouse, University of Dundee), and Tubulin (1:2000, Abcam, ab6160) for 1 h at room temperature.

Techniques: Suspension, Incubation, Staining, Expressing, Mutagenesis, Knockdown, Western Blot, Immunofluorescence

Journal: Oncotarget

Article Title: Human cancer cells utilize mitotic DNA synthesis to resist replication stress at telomeres regardless of their telomere maintenance mechanism

doi: 10.18632/oncotarget.24745

Figure Lengend Snippet: ( A ) Representative images EdU (red) incorporation at telomeres (marked by telomeric FISH; green) on metaphase chromosomes (stained with DAPI; blue) in control, SLX4-, RAD52-, or MUS81-depleted U2OS, as indicated. ( B – D ) Quantification of total EdU incorporation (combined telomeric and non-telomeric) and EdU incorporation specifically at telomeres in U2OS cells. ( E ) Quantification of EdU incorporation specifically at telomeres in HeLa-LT cells. EdU was quantified on metaphase chromosomes following the indicated siRNA depletions. Data represent the means of at least three independent experiments. Error bars indicate SEM. See also .

Article Snippet: The primary antibodies and their dilutions for Western blotting analysis were RAD52 (1:100 Santa Cruz sc-8350, or 1:500 Abcam ab103067), SLX4 (1:200, S714C raised against the residues 1535-1834, a gift from Dr. J.

Techniques: Staining

Journal: Nucleic Acids Research

Article Title: SLX4IP acts with SLX4 and XPF–ERCC1 to promote interstrand crosslink repair

doi: 10.1093/nar/gkz769

Figure Lengend Snippet: SLX4IP is involved in ICL repair. ( A ) Results of clonogenic survival assays conducted with HEK293A-WT and SLX4IP-KO cells exposed to DNA damage-inducing agents (mitomycin C [MMC], campothecin [CPT], ultraviolet [UV] radiation, or ionizing radiation [IR]). For each cell line, the viability of untreated cells was defined as 100%. Data are presented as the mean ± standard error of the mean (SEM; n = 3). ( B and C ) HEK293A-WT, SLX4IP-KO, XPF-KO and MUS81-KO cells were treated with the indicated concentrations of MMC for 24 h or were nontreated (NT). Cells were collected and fixed with 70% ethanol before fluorescence-activated cell sorting (FACS) analysis of the cell-cycle distribution under different treatment conditions. The mean percentages of cells in the G2/M phase from three independent repeats of FACS are shown. The results were compared statistically with those for untreated cells. Statistical analysis was performed using the Student's t -test. ** P < 0.01, *** P < 0.001, ns: not significant. P values <0.05 were considered statistically significant. ( D ) Immunostainings of SLX4, SLX4IP and γH2AX proteins performed following UV laser-induced DNA damage with the indicated antibodies. KO, knockout; WT, wild type.

Article Snippet: In this study, antibodies against the following proteins were used for western blotting and/or immunostaining: SLX4 (A302-270A; Bethyl Laboratories, Montgomery, TX, USA) or an antibody developed in-house against the antigen comprising SLX4 231-460 , XPF (A301-315A; Bethyl Laboratories), MUS81 (sc-53382; Santa Cruz Biotechnology, Dallas, TX, USA), SLX1 (ab182501; Abcam, Cambridge, UK), MSH2 (2017S; Cell Signaling Technology, Danvers, MA), SLX4IP (an antibody developed in-house against the antigen comprising SLX4IP 205-408 ), γH2AX (2577S; Cell Signaling Technology), Flag M2 (F3165-5MG; Sigma-Aldrich, St. Louis, MO, USA), α-tubulin (T6199-200UL; Sigma-Aldrich), vinculin (V9131; Sigma-Aldrich), MBP (sc-13564; Santa Cruz Biotechnology), GST (sc-138; Santa Cruz Biotechnology), ERCC1 (sc-17809; Santa Cruz Biotechnology), phospho-Chk2 (Thr68, 2661S; Cell Signaling Technology), Chk2 (2662S; Cell Signaling Technology), phospho-Chk1 (Ser345, 2348S; Cell Signaling Technology), and Chk1 (2360S; Cell Signaling Technology).

Techniques: Fluorescence, FACS, Knock-Out

Journal: Nucleic Acids Research

Article Title: SLX4IP acts with SLX4 and XPF–ERCC1 to promote interstrand crosslink repair

doi: 10.1093/nar/gkz769

Figure Lengend Snippet: The interaction between SLX4IP and SLX4 is coordinated with XPF. ( A ) Tandem affinity purification and mass spectrometry (TAP-MS) was conducted to identify SLX4IP-interacting proteins. Lists of high-confidence candidate interacting proteins from mass spectrometry analysis of cells treated with or without mitomycin C (MMC) are presented. ( B ) HEK293A cells were transfected with constructs encoding SLX4-SFB or its deletion variants and were subjected to immunoprecipitation (IP) with S beads. Western blotting was conducted with antibodies as indicated. ( C ) Experiments were done as in B, except that SLX4-SFB wild type and SLX4 m -SFB (L530A, F545A, Y546A and L550A) variants were subjected to precipitation with S beads. FL, full length (controls); PSM, peptide spectrum match.

Article Snippet: In this study, antibodies against the following proteins were used for western blotting and/or immunostaining: SLX4 (A302-270A; Bethyl Laboratories, Montgomery, TX, USA) or an antibody developed in-house against the antigen comprising SLX4 231-460 , XPF (A301-315A; Bethyl Laboratories), MUS81 (sc-53382; Santa Cruz Biotechnology, Dallas, TX, USA), SLX1 (ab182501; Abcam, Cambridge, UK), MSH2 (2017S; Cell Signaling Technology, Danvers, MA), SLX4IP (an antibody developed in-house against the antigen comprising SLX4IP 205-408 ), γH2AX (2577S; Cell Signaling Technology), Flag M2 (F3165-5MG; Sigma-Aldrich, St. Louis, MO, USA), α-tubulin (T6199-200UL; Sigma-Aldrich), vinculin (V9131; Sigma-Aldrich), MBP (sc-13564; Santa Cruz Biotechnology), GST (sc-138; Santa Cruz Biotechnology), ERCC1 (sc-17809; Santa Cruz Biotechnology), phospho-Chk2 (Thr68, 2661S; Cell Signaling Technology), Chk2 (2662S; Cell Signaling Technology), phospho-Chk1 (Ser345, 2348S; Cell Signaling Technology), and Chk1 (2360S; Cell Signaling Technology).

Techniques: Affinity Purification, Mass Spectrometry, Transfection, Construct, Immunoprecipitation, Western Blot

Journal: Nucleic Acids Research

Article Title: SLX4IP acts with SLX4 and XPF–ERCC1 to promote interstrand crosslink repair

doi: 10.1093/nar/gkz769

Figure Lengend Snippet: The conserved N-terminus of SLX4IP is responsible for its interaction with SLX4–XPF–ERCC1. ( A ) HEK293A cells were transfected with constructs encoding SLX4IP-SFB or its deletion variants and subjected to immunoprecipitation (IP) pulldown with S beads. Western blotting conducted with antibodies as indicated. A schematic overview of the deletion strategy and protein-protein interaction results is presented. ( B ) Conservation of sequences at the N-terminus of SLX4IP. The alignment of the N-terminal sequences of SLX4IP in humans, mice, zebrafish and Xenopus , established using the Clustal Omega sequence alignment program, is shown. ( C ) HEK293A cells were transfected with constructs encoding SLX4IP-SFB or its point mutation variants and subjected to IP with S beads. Western blotting was conducted with antibodies as indicated. ( D ) SLX4 was subjected to IP with anti-SLX4 serum from HEK293A-WT or SLX4IP-KO cells (clone #6 and clone #13). Western blotting was conducted with antibodies as indicated. ( E ) HEK293A-WT or SLX4IP-KO cells were transfected with SFB-XPF and subjected to IP with S beads. Western blotting was conducted with antibodies as indicated. ( F ) Western blots detecting the interaction between XPF and SLX4 in HEK293A-WT and SLX4IP-KO cells were analyzed by ImageJ. The mean relative interaction from five experiments is shown. FL, full length (controls); KO, knockout; WT, wild type.

Article Snippet: In this study, antibodies against the following proteins were used for western blotting and/or immunostaining: SLX4 (A302-270A; Bethyl Laboratories, Montgomery, TX, USA) or an antibody developed in-house against the antigen comprising SLX4 231-460 , XPF (A301-315A; Bethyl Laboratories), MUS81 (sc-53382; Santa Cruz Biotechnology, Dallas, TX, USA), SLX1 (ab182501; Abcam, Cambridge, UK), MSH2 (2017S; Cell Signaling Technology, Danvers, MA), SLX4IP (an antibody developed in-house against the antigen comprising SLX4IP 205-408 ), γH2AX (2577S; Cell Signaling Technology), Flag M2 (F3165-5MG; Sigma-Aldrich, St. Louis, MO, USA), α-tubulin (T6199-200UL; Sigma-Aldrich), vinculin (V9131; Sigma-Aldrich), MBP (sc-13564; Santa Cruz Biotechnology), GST (sc-138; Santa Cruz Biotechnology), ERCC1 (sc-17809; Santa Cruz Biotechnology), phospho-Chk2 (Thr68, 2661S; Cell Signaling Technology), Chk2 (2662S; Cell Signaling Technology), phospho-Chk1 (Ser345, 2348S; Cell Signaling Technology), and Chk1 (2360S; Cell Signaling Technology).

Techniques: Transfection, Construct, Immunoprecipitation, Western Blot, Sequencing, Mutagenesis, Knock-Out

Journal: Nucleic Acids Research

Article Title: SLX4IP acts with SLX4 and XPF–ERCC1 to promote interstrand crosslink repair

doi: 10.1093/nar/gkz769

Figure Lengend Snippet: The helicase-like domain of XPF is responsible for its interaction with SLX4 and SLX4IP. ( A ) HEK293A-WT or XPF-KO cells were transfected with SLX4IP-SFB and subjected to immunoprecipitation (IP) with S beads. Western blotting was conducted with antibodies as indicated. ( B ) A schematic overview of the XPF domains, XPF deletion mutants, and results of protein-protein interactions is presented. ( C and D ) XPF-KO cells were transfected with a construct encoding SFB-XPF, a deletion mutant of SFB-XPF, or a point mutation variant of SFB-XPF along with a construct encoding GFP-SLX4 ( C ) or GFP-SLX4IP ( D ). Cell lysates were subjected to IP with S beads. Western blotting was conducted with antibodies as indicated. ( E ) A schematic model shows the interaction domain of SLX4–XPF–ERCC1-SLX4IP. ( F ) To induce degradation of SLX4-mAID-SFB protein, 1 mM auxin (IAA) was added to cells for 48 h. Western blotting was conducted with antibodies as indicated. ( G ) HEK293A SLX4-mAID-SFB knock-in (KI) cells were infected with a virus encoding HA-SLX4IP and treated with or without auxin for 48 h. Cells were collected, lysed, and subjected to IP with hemagglutinin (HA) beads. Western blotting was conducted with antibodies as indicated. ( H ) HEK293A cells were infected with a virus encoding inducible control shRNA or shRNA for SLX4 (#84 and #85). To induce knockdown of SLX4 protein, 1 μg/ml of doxycycline (DOX) was added to cells for 48 h. Western blotting was conducted to detect the levels of the indicated proteins in whole-cell extract and chromatin extracts. ( I ) The expression levels of proteins in HEK293A-WT, SLX4IP-KO, XPF-KO and XPF/SLX4IP-DKO cells were detected with antibodies as indicated. HLD, helicase-like domain; KO, knockout; WT, wild type.

Article Snippet: In this study, antibodies against the following proteins were used for western blotting and/or immunostaining: SLX4 (A302-270A; Bethyl Laboratories, Montgomery, TX, USA) or an antibody developed in-house against the antigen comprising SLX4 231-460 , XPF (A301-315A; Bethyl Laboratories), MUS81 (sc-53382; Santa Cruz Biotechnology, Dallas, TX, USA), SLX1 (ab182501; Abcam, Cambridge, UK), MSH2 (2017S; Cell Signaling Technology, Danvers, MA), SLX4IP (an antibody developed in-house against the antigen comprising SLX4IP 205-408 ), γH2AX (2577S; Cell Signaling Technology), Flag M2 (F3165-5MG; Sigma-Aldrich, St. Louis, MO, USA), α-tubulin (T6199-200UL; Sigma-Aldrich), vinculin (V9131; Sigma-Aldrich), MBP (sc-13564; Santa Cruz Biotechnology), GST (sc-138; Santa Cruz Biotechnology), ERCC1 (sc-17809; Santa Cruz Biotechnology), phospho-Chk2 (Thr68, 2661S; Cell Signaling Technology), Chk2 (2662S; Cell Signaling Technology), phospho-Chk1 (Ser345, 2348S; Cell Signaling Technology), and Chk1 (2360S; Cell Signaling Technology).

Techniques: Transfection, Immunoprecipitation, Western Blot, Protein-Protein interactions, Construct, Mutagenesis, Variant Assay, Knock-In, Infection, Virus, Control, shRNA, Knockdown, Expressing, Knock-Out

Journal: Nucleic Acids Research

Article Title: SLX4IP acts with SLX4 and XPF–ERCC1 to promote interstrand crosslink repair

doi: 10.1093/nar/gkz769

Figure Lengend Snippet: SLX4IP functions in the same pathway as XPF but not as MUS81. ( A–C ) Proliferation of HEK293A-WT, SLX4IP-KO, XPF-KO, and other cell lines as indicated was measured using a CellTiter-Glo assay after 3 days in the presence of the indicated concentrations of mitomycin C (MMC). Data are presented as mean ± the standard error of the mean ( n = 3). ( D ) Experiments were done as presented in A, B, and C. Proliferation of the indicated cell lines was measured using a CellTiter-Glo assay after 3 days in the presence of the indicated concentrations of camptothecin (CPT). DKO, double knockout; IC 50 , half-maximal inhibitory concentration; KO, knockout; WT, wild type.

Article Snippet: In this study, antibodies against the following proteins were used for western blotting and/or immunostaining: SLX4 (A302-270A; Bethyl Laboratories, Montgomery, TX, USA) or an antibody developed in-house against the antigen comprising SLX4 231-460 , XPF (A301-315A; Bethyl Laboratories), MUS81 (sc-53382; Santa Cruz Biotechnology, Dallas, TX, USA), SLX1 (ab182501; Abcam, Cambridge, UK), MSH2 (2017S; Cell Signaling Technology, Danvers, MA), SLX4IP (an antibody developed in-house against the antigen comprising SLX4IP 205-408 ), γH2AX (2577S; Cell Signaling Technology), Flag M2 (F3165-5MG; Sigma-Aldrich, St. Louis, MO, USA), α-tubulin (T6199-200UL; Sigma-Aldrich), vinculin (V9131; Sigma-Aldrich), MBP (sc-13564; Santa Cruz Biotechnology), GST (sc-138; Santa Cruz Biotechnology), ERCC1 (sc-17809; Santa Cruz Biotechnology), phospho-Chk2 (Thr68, 2661S; Cell Signaling Technology), Chk2 (2662S; Cell Signaling Technology), phospho-Chk1 (Ser345, 2348S; Cell Signaling Technology), and Chk1 (2360S; Cell Signaling Technology).

Techniques: Glo Assay, Double Knockout, Concentration Assay, Knock-Out

Journal: Nucleic Acids Research

Article Title: SLX4IP acts with SLX4 and XPF–ERCC1 to promote interstrand crosslink repair

doi: 10.1093/nar/gkz769

Figure Lengend Snippet: SLX4IP stabilizes the interaction between SLX4 and XPF–ERCC1, especially after DNA damage. ( A ) HEK293A-WT and SLX4IP-KO cells were nontreated (NT) or treated with 1 μm mitomycin C (MMC) for 24 h, and radiated with 100 J/m 2 ultraviolet (UV) radiation and released for 4 h. Whole-cell extracts were analyzed by gel filtration on a Superdex 200 10/300 GL preparative-grade column. 96 fractions were collected in volumes of 200 μl. Fractions as indicated were analyzed with antibodies as indicated. ( B ) SLX4 was subjected to immunoprecipitation (IP) with anti-SLX4 serum using lysates of HEK293A-WT cells, XPF-KO cells, and other indicated cell lines. ( C ) The same amount of GFP or GFP-SLX4 293-638 was mixed with GFP beads. After rotation in a cold room for 1 h, protein-bound beads were washed with NETN lysis buffer. The indicated amounts of XPF and/or SLX4IP were mixed with NETN lysis buffer to the same volume, and the mixture was allowed to bind to protein bound on GFP beads. After mixing in a cold room for 1 h, beads were thoroughly washed and boiled at 95°C in Laemmli buffer. GFP and GFP-SLX4 293-638 were detected using Coomassie Blue staining. XPF was detected using an anti-GST antibody, and SLX4IP was detected using an anti-MBP antibody. Quantification analysis was done with ImageJ and the amount of relative XPF protein bound to the beads is shown. DKO, double knockout; KO, knockout; WT, wild type.

Article Snippet: In this study, antibodies against the following proteins were used for western blotting and/or immunostaining: SLX4 (A302-270A; Bethyl Laboratories, Montgomery, TX, USA) or an antibody developed in-house against the antigen comprising SLX4 231-460 , XPF (A301-315A; Bethyl Laboratories), MUS81 (sc-53382; Santa Cruz Biotechnology, Dallas, TX, USA), SLX1 (ab182501; Abcam, Cambridge, UK), MSH2 (2017S; Cell Signaling Technology, Danvers, MA), SLX4IP (an antibody developed in-house against the antigen comprising SLX4IP 205-408 ), γH2AX (2577S; Cell Signaling Technology), Flag M2 (F3165-5MG; Sigma-Aldrich, St. Louis, MO, USA), α-tubulin (T6199-200UL; Sigma-Aldrich), vinculin (V9131; Sigma-Aldrich), MBP (sc-13564; Santa Cruz Biotechnology), GST (sc-138; Santa Cruz Biotechnology), ERCC1 (sc-17809; Santa Cruz Biotechnology), phospho-Chk2 (Thr68, 2661S; Cell Signaling Technology), Chk2 (2662S; Cell Signaling Technology), phospho-Chk1 (Ser345, 2348S; Cell Signaling Technology), and Chk1 (2360S; Cell Signaling Technology).

Techniques: Filtration, Immunoprecipitation, Lysis, Staining, Double Knockout, Knock-Out

Journal: Nucleic Acids Research

Article Title: SLX4IP acts with SLX4 and XPF–ERCC1 to promote interstrand crosslink repair

doi: 10.1093/nar/gkz769

Figure Lengend Snippet: Loss of SLX4IP reduces the repair efficiency of interstrand crosslink repair in vivo. (A) Working model of SLX4–XPF–ERCC1-SLX4IP interaction and function. Our current working hypothesis is that SLX4IP acts to promote the interaction between SLX4 and XPF–ERCC1, especially following DNA damage. This function of SLX4IP is critical for interstrand crosslink (ICL) repair. In our XPF-KO cells, truncated XPF was expressed only at low levels and SLX4IP protein was mostly degraded. Therefore, a very limited SLX4–XPF–ERCC1-SLX4IP complex formed and we observed severe ICL repair defects. When we overexpressed SLX4IP in these cells, the interaction between truncated XPF and SLX4 was enhanced, which suppressed the hypersensitivity of XPF-KO or XPF/SLX4IP-DKO cells to mitomycin C (MMC). We found that mAID-mediated downregulation of SLX4 also caused downregulation of SLX4IP protein, which may result in the failure of XPF–ERCC1 recruitment and diminished cell survival. As for SLX4IP-KO cells, SLX4 could still interact with XPF–ERCC1, but the complex was not stable; this was especially true following DNA damage, which resulted in reduced ICL repair efficiency. ( B ) Cells were treated with 1 μm MMC for 1 h and released for the indicated times. Samples were taken to detect the unhooking of ICL using a modified comet assay (please see the Material and Methods section for detailed description). DNA ICL is expressed as the percentage of decrease in Olive tail movement. Data are presented as the mean ± the standard error of the mean ( n = 3). ( C ) HEK293A cells were mock-treated (Mock) or treated with MMC (either 1# 0.5 μg/ml MMC for 1 h and release for 24 h, or 2# 0.1 μg/ml MMC for 24 h). After treatment, whole-cell extracts were prepared and subjected to Western blotting with the indicated antibodies. ( D ) HEK293A-WT, SLX4IP-KO, XPF-KO, and MUS81-KO cells were exposed to 0.5 μg/ml MMC for 1 h and allowed to recover after removal of the drug. Cells were then fixed with 3% paraformaldehyde and stained with an anti-γH2AX antibody. The percentages of cells with more than five γH2AX foci are shown. Data are presented as the mean ± the standard error of the mean ( n = 3). Statistical analysis was performed using the Student's t -test. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant. P values less than 0.05 were considered statistically significant. KO, knockout; WT, wild type.

Article Snippet: In this study, antibodies against the following proteins were used for western blotting and/or immunostaining: SLX4 (A302-270A; Bethyl Laboratories, Montgomery, TX, USA) or an antibody developed in-house against the antigen comprising SLX4 231-460 , XPF (A301-315A; Bethyl Laboratories), MUS81 (sc-53382; Santa Cruz Biotechnology, Dallas, TX, USA), SLX1 (ab182501; Abcam, Cambridge, UK), MSH2 (2017S; Cell Signaling Technology, Danvers, MA), SLX4IP (an antibody developed in-house against the antigen comprising SLX4IP 205-408 ), γH2AX (2577S; Cell Signaling Technology), Flag M2 (F3165-5MG; Sigma-Aldrich, St. Louis, MO, USA), α-tubulin (T6199-200UL; Sigma-Aldrich), vinculin (V9131; Sigma-Aldrich), MBP (sc-13564; Santa Cruz Biotechnology), GST (sc-138; Santa Cruz Biotechnology), ERCC1 (sc-17809; Santa Cruz Biotechnology), phospho-Chk2 (Thr68, 2661S; Cell Signaling Technology), Chk2 (2662S; Cell Signaling Technology), phospho-Chk1 (Ser345, 2348S; Cell Signaling Technology), and Chk1 (2360S; Cell Signaling Technology).

Techniques: In Vivo, Modification, Single Cell Gel Electrophoresis, Western Blot, Staining, Knock-Out