Fig 2: 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.

Fig 3: 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.

Fig 4: 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/m2 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-SLX4293-638 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-SLX4293-638 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.

Fig 5: 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 SLX4m-SFB (L530A, F545A, Y546A and L550A) variants were subjected to precipitation with S beads. FL, full length (controls); PSM, peptide spectrum match.