Fig 2: RNF168 and the ubiquitylation sites of SLFN11 in its middle domain promotes chromatin recruitment of SLFN11(A) Representative immunofluorescence images showing chromatin-bound SLFN11 (green), RNF168 (red) and DAPI (blue). DOX-inducible SLFN11-expressing U2OS cells were treated with VLX-1570 (1 μM) or CPT (10 μM) for 2 hrs after DOX induction (72 hrs) and siRNF168 transfection (48 hrs). Scale bar: 5 μm.(B, C) Quantification of chromatin-bound SLFN11 and RNF168 signals in individual cells for the indicated treatments (n = 57 – 97 cells per condition). **p < 0.01. ***p < 0.001. ****p < 0.0001 (two-tailed unpaired t test). a.u., arbitrary units.(D) Representative immunofluorescence images showing chromatin-bound SLFN11 (magenta) and DAPI (blue) in DOX-inducible SLFN11 clones (WT, 3KR) of U2OS cells. Cells were treated with VLX-1570 (1 μM) or CPT (10 μM) for 2 hrs. Scale bar: 10 μm.(E) Quantification of chromatin-bound SLFN11 fluorescence (average per condition) for the treatment depicted in panel D (mean ± SEM, N = 411 – 690 cells per condition). ***p < 0.0001 (two-tailed unpaired t test).(F) SLFN11 immunoblots of chromatin fraction. DOX-inducible SLFN11 clones (WT, 3KR) of U2OS cells were treated with VLX-1570 (5 μM, 10 μM, 2 hrs) after the DOX induction (72 hrs), lysed and fractionated with pre-extraction buffer.

Fig 3: SLFN11 chromatin recruitment by DNA damage is dependent on both ubiquitin and RPA(A) SLFN11 chromatin recruitment by VLX-1570 and camptothecin (CPT). Representative immunofluorescence images show chromatin-bound SLFN11 (green), RPA2 (red), and DAPI (blue). DU145 cells were treated with VLX-1570 (1 μM) or CPT (10 μM) for 4 hrs. Scale bar: 10μm.(B) VLX-1570 recruits SLFN11 in an RPA-independent manner. Left: Representative immunofluorescence images showing chromatin-bound SLFN11 (green), RPA2 (red), and DAPI (blue). Scale bar: 10 μm. DOX-induced SLFN11-expressing U2OS cells were treated with VLX-1570 (1 μM) or CPT (10 μM) for 4 hrs. Right: Tracing of the distribution of signals along the white dashed lines shown in the left panels.(C) Representative immunofluorescence images showing chromatin-bound SLFN11 (green), γH2AX (red) and DAPI (blue). DOX-inducible SLFN11-expressing U2OS cells were treated with VLX-1570 (1 μM) or CPT (10 μM) for 4 hours. Scale bar: 10μm.(D, E) Quantification of chromatin-bound SLFN11 and γH2AX signals in individual cells for the indicated treatments. (n = 118 – 166 cells per condition) *p < 0.05, **p < 0.01 **p < 0.001. ****p < 0.0001 (two-tailed unpaired t test). a.u., arbitrary units.

Fig 4: SLFN11 interacts with the E3 ubiquitin ligase RNF168 which targets the lysine residues in the middle linker domain of SLFN11(A) Immunoprecipitation of RNF168 with SLFN11. HEK293 cells were transfected with Flag-tagged SLFN11 for 48 hrs. Cell lysates were immunoprecipitated with anti-flag M2 agarose beads. RNF168 was probed with anti-RNF168 antibody.(B) Immunoprecipitation of SLFN11 with RNF168. HEK293 cells were transfected with HA-tagged RNF168 for 48 hrs. Cell lysate were immunoprecipitated with anti-HA agarose beads. SLFN11 was probed with anti-SLFN11 antibody.(C) SLFN11 and RNF168 immunoblots of whole-cell lysate showing that SLFN11 expression does not affect RNF168 expression. DOX-inducible SLFN11-expressing U2OS cells were lysed after the DOX induction (72 hrs).(D) Immunoprecipitation of SLFN11 with an antibody against endogenous RNF168. After DOX induction for 72 hrs, U2OS cells were lysed and immunoprecipitated with anti-RNF168 with protein A/G agarose beads. SLFN11 was probed with anti-Flag M2 antibody.(E) Representative immunofluorescence images of proximity ligation assays (PLA) of SLFN11 and RNF168. DOX-inducible SLFN11-expressing U2OS cells were treated for 24 hrs with DOX and then incubated with primary antibodies against SLFN11 and RNF168.(F) Quantification of the PLA as in panel C. Points denote the mean fluorescence intensity (MFI) of PLA/cell with ± SEM (n > 80). ****p < 0.0001 (two-tailed unpaired t test).(G) RNF168-dependent ubiquitylation of SLFN11. HEK293 cells were transfected with His-Ub and siRNF168 mix. Cells were lysed and immunoprecipitated with Ni-NTA beads. Ubiquitylated SLFN11 [SLFN11-(Ub)n] was detected with anti-SLFN11 antibody.(H) Ubiquitylation of SLFN11 by RNF168. 293T cells were transfected with SLFN11-Flag, HA-RNF168, and His-Ub. Cells were lysed and immunoprecipitated with Ni-NTA beads. Ubiquitylated SLFN11 was detected with anti-Flag antibody.(I) K27-dependent ubiquitylation of SLFN11 by RNF168. 293T cells were transfected with SLFN11-Flag, HA-RNF168, and His-Ub (WT, K27R, K48R, and K63R). Cells were lysed and immunoprecipitated with Ni-NTA beads. Ubiquitylated SLFN11 was detected with anti-Flag antibody.(J) Mapping of the cellular ubiquitylation sites of SLFN11 by RNF168. Top: schematic representation of the tested lysine residues as potential targets for ubiquitylation by RNF-168 HEK293T cells were transfected with SLFN11-Flag, SLFN11-Flag K/R mutants, HA-RNF168, and His-Ub as indicated. Cells were lysed and immunoprecipitated with Ni-NTA beads. Ubiquitylated SLFN11 was probed with anti-Flag antibody.(K) Schematic representation of the location of the ubiquitylated lysine residues of SLFN11 (K390/K391, K429, K743, and K890) mapped onto SLFN11 protein structure (PDB: 7ZEL).(L) Schematic representation of the constructs used for the experiments shown in panel M.(M) Ubiquitylation sites of SLFN11 in its linker domain by RNF168. HEK293T cells were transfected with SLFN11-Flag, SLFN11-Flag K/R mutants (3KR:K390R/K391R/K429R, 2KR: K429R/K743R, as indicated), HA-RNF168, and His-Ub. Cells were lysed and immunoprecipitated with Ni-NTA beads. Ubiquitylated SLFN11 was probed with anti-Flag antibody.

Fig 5: SLFN11 chromatin binding induced VLX-1570 localizes in open chromatin at promoters and suppresses RNA synthesis(A) Left: Representative immunofluorescence images showing the colocalization of chromatin-bound SLFN11 (green) and H3K27ac (red). Nuclei are stained with DAPI (blue). DOX-induced SLFN11-expressing U2OS cells were treated with VLX-1570 (1 μM) for 4 hrs. Right: Quantification of chromatin-bound SLFN11 and H3K27ac in individual cells for the indicated treatments (n = 228 – 327 cells per condition). **p < 0.01, ns, non-significant, ***p < 0.001, ****p < 0.0001 (one-way ANOVA). a.u., arbitrary units(B) Left: Representative immunofluorescence images showing SLFN11-dependent inhibition of EU incorporation (green) by VLX-1570. Scale bar: 20 μm. DOX-inducible SLFN11-expressing U2OS cells were treated with VLX-1570 (1 μM). Nuclei are stained with DAPI (blue). Right: Quantification of EU in individual cells. (n = 109 – 166 cells per condition). **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA) a.u.., arbitrary units(C, D) Pie charts depicting the genomic distribution of SLFN11-Flag (C) and H3K27ac (D) ChIP-seq peaks across annotated genomic features in the presence or absence of VLX-1570.(E, F, and G) Heatmaps and average signal profiles of SLFN11-Flag ChIP-seq (E), H3K27ac ChIP-seq (F), and SLFN11-Flag signal centered on H3K27ac peaks (G) with or without VLX-1570 treatment.(H) Venn diagram showing the number of SLFN11-Flag peaks, H3K27ac peaks, and their overlap identified in the indicated ChIP-seq analyses. 68.4% and 74.1% of the SLFN11 peaks coincide with the H3K27 peaks in the absence and presence of VLX-1570, respectively.(I) Genome browser tracks illustrating ChIP-seq signal enrichment at the FOS promoter for SLFN11-Flag and H3K27ac in the presence or absence of VLX-1570.