Journal: Nature Communications

Article Title: ERCC6L2 ensures repair fidelity for staggered-end DNA double-strand breaks

doi: 10.1038/s41467-026-69843-w

Figure Lengend Snippet: a Competition assay monitoring the ratio of RPE1 TP53 −/− cells expressing gRNA targeting LacZ vs expressing gRNA targeting ERCC6L2 after etoposide treatment (mean of n = 5 biological replicates ± SD). WT, wild-type. b Quantification of RPA32 positive RPE1 TP53 −/− cells by cell cycle status. Cells were treated for 30 min with 10 nM ATMi (AZD0156), then cells were treated with 100 µM of etoposide + 10 nM ATMi for 1 h (mean of n = 3 biological replicates ± SD). c Scheme for a genome-wide CRISPR knockout screen in RPE1 TP53 −/− (IC 20 = 120 nM, published in ref. ) and TP53 −/− ; ERCC6L2 −/− backgrounds (IC 20 = 45 nM). d Rank plots from normZ effect scores of etoposide vs DMSO of CRISPR knockout screens. e Clonogenic survival assay in RPE1 TP53 −/− titrated with etoposide (mean of n = 3 biological replicates ± SD). f Clonogenic survival assay in RPE1 TP53 −/− cells transfected with the indicated siRNAs and titrated with etoposide (mean of n = 3 biological replicates ± SD). g Quantification of RPA32 foci in RPE1 TP53 −/− cells treated with etoposide. Nuclei showing more than 5 RPA foci were selected (mean of n = 3 biological replicates). h Quantification of long-read sequencing reads with deletion lengths greater than 1 kb in RPE1 TP53 −/− cells treated with either DMSO or MRE11 inhibitor (MRE11i) ( n = 1 biological replicate). i Quantification of PLA signal intensity for NBS1-ɣH2AX in RPE1 TP53 −/− cells treated with etoposide (mean of n = 3 biological replicates). j Quantification of PLA foci for MRE11-CtIP in etoposide-treated RPE1 TP53 −/− cells (mean of n = 2 technical replicates). k MRE11 peak profile surrounding each Cas12a gMulti target site in RPE1 TP53 −/− cells. l Normalized quantification of the area under the curve (AUC) of MRE11 at each of the 27 Cas12a gMulti target sites (1 biological replicate; mean ± SD). Statistical significance was determined by two-way ANOVA and Tukey’s multiple comparison test in ( a, b, e , f ); one-way ANOVA and Tukey’s multiple comparison test in ( g ); two-tailed Welch’s t-test in ( l ). Source data are provided as a file.

Article Snippet: The isolated DNA was sonicated to obtain ~300 bp chromatin fragments using a Covaris S2 with the following settings: 12 cycles of duty cycle 5%, intensity 5, 200 cycles per burst for 60 s. Ten micrograms of MRE11 antibody (NBP3-25349; Novus Biologicals) per ChIP were prebound to protein A Dynabeads (Invitrogen).

Techniques: Competitive Binding Assay, Expressing, Genome Wide, CRISPR, Knock-Out, Clonogenic Cell Survival Assay, Transfection, Sequencing, Comparison, Two Tailed Test