Fig 2: 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.

Fig 3: SLX4IP Interacts with BLM Helicase(A) U2OS cells transfected with the indicated siRNAs were fixed and processed for BLM immunofluorescence followed by telomeric PNA (TelG) FISH. Scale bar represents 10 µm. Dashed lines indicate nucleus outlines (as determined using DAPI staining; not shown). Insets represent 3× magnifications of the indicated fields.(B) Quantification of (A). At least 100 cells per condition were counted. Data are presented as 5th–95th percentiles; n = 3; ****p < 0.00001, one-way ANOVA; ns, not significant.(C) U2OS whole-cell extracts were separated using SDS-PAGE and analyzed using BLM immunoblotting. Tubulin was used as loading control. Numbers on the right denote molecular weight (kDa). Numbers below indicate protein levels. Protein levels were normalized to SLX4IP+/+, which was arbitrarily assigned a value of 1.(D) Whole-cell extracts from HEK293 cells transiently expressing GFP constructs were subjected to GFP-trap co-immunoprecipitation (IP). Input and IP samples were separated using SDS-PAGE and analyzed using GFP, SLX4IP, and RMI2 immunoblotting. Numbers denote molecular weight (kDa).(E) Whole-cell extracts from HEK293 cells transiently expressing GFP constructs were subjected to GFP-trap co-immunoprecipitation (IP). Input and IP samples were separated using SDS-PAGE and analyzed using GFP and BLM immunoblotting. Numbers denote molecular weight (kDa).(F) Whole-cell extracts from HEK293 cells transfected with the indicated siRNAs and transiently expressing GFP constructs were subjected to GFP-trap co-immunoprecipitation (IP). Input and IP samples were separated using SDS-PAGE and analyzed using GFP, SLX4, BLM, and XPF immunoblotting. Tubulin was used as loading control. Numbers denote molecular weight (kDa).(G) Recombinant Flag-BLM and SLX4IP proteins were subjected to BLM co-immunoprecipitation (IP). Normal IgGs were used as negative IP control. Input and IP samples were separated using SDS-PAGE and analyzed using BLM and SLX4IP immunoblotting. Numbers denote molecular weight (kDa).See also Figure S7.

Fig 4: 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 (46,82). (B) HeLa FITo cells were transfected with expression vectors coding for GFP, FLAG-SLX4, GFP-MSH2 WT and/or GFP-MSH2M453I, 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.

Fig 5: T-circle formation in MSK-41 cells is dependent on SLX4.(A) Two shRNAs (SLX4-1 and SLX4-2) were used to knockdown SLX4 expression. (B) T-circle formation was measured in the MSK-41 SLX4 knockdown strains relative to MSK-41 with a control shRNA. Mean and standard deviation were calculated from two independent experiments. *P<0.05, **P<0.01 by unpaired two-tailed t-test.