Extended Data Fig. 9: Knockout of the Fanconi anaemia and NEIL3 pathways have additive effects on ICL sensitivity in mammalian cells.

a, Immunoblot analysis of NEIL3 expression in wild-type, NEIL3-knockout and FANCL/NEIL3-knockout HAP1 cell lines. Histone H3 was detected as a loading control. Non-specifically detected proteins are marked with asterisks. Analysis performed twice. b, Schematic of FANCL targeting by CRISPR. (i) Human FANCL exon 10, single-guide (sg)RNA binding sites, and homology arm targets; (ii) FANCL-Puro targeting construct with homology arms flanking exon 10; (iii) Targeted FANCL allele with integrated puromycin-resistance cassette. c, Detection of the integrated puromycin-resistance cassette in HAP1 cells by FANCL long-range PCR. Analysis performed twice. d, Analysis of FANCD2 ubiquitylation in mitomycin-C-treated wild-type, FANCL-knockout and FANCL/NEIL3-knockout HAP1 cell lines to confirm FANCL knockout. Vinculin was detected as a loading control. FANCL is the catalytic subunit of the Fanconi anaemia core complex, which ubiquitylates FANCD2. Analysis performed twice. e, Neil3 qRT–PCR confirming gene disruption in CH12 cell lines. ND, not detected. Analysis performed once. f, Analysis of FANCD2 ubiquitylation in mitomycin-C-treated Fancb-knockout and Neil3/Fancb-knockout CH12 cell lines to confirm Fancb knockout. Vinculin was detected as a loading control. A non-specifically detected protein is marked with an asterisk. Analysis performed once. g, Cell viability of wild-type, Fancb-knockout, Neil3-knockout or Fancb/Neil3-knockout CH12 cells after exposure to trioxsalen and UVA irradiation. Two independent clones were used for the single mutants and three independent clones were used for the double mutant. Data are mean ± s.e.m. We speculate that, relative to HAP1 cells, CH12 cells may be more reliant on the Fanconi anaemia pathway to repair trioxsalen-induced damage owing to lower expression levels of NEIL3. Three independent experiments.