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

a, Immunoblot analysis of NEIL3 expression in wild-type, NEIL3, and FANCL/NEIL3 knockout HAP1 cell lines. Histone H3 was detected as a loading control. Non-specifically detected proteins are marked with asterisks.

b, Schematic of FANCL CRISPR targeting. (i) Human FANCL exon 10, sgRNA 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.

d, Analysis of FANCD2 ubiquitylation in MMC-treated wild-type, FANCL, 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 FA core complex, which ubiquitylates FANCD2.

e, Neil3 qRT-PCR confirming gene disruption in CH12 cell lines. ND, not detected.

f, Analysis of FANCD2 ubiquitylation in mitomycin C-treated CH12 Fancb single and Neil3/Fancb double knockout cell lines to confirm Fancb knockout. Vinculin was detected as a loading control. A non-specifically detected protein is marked with an asterisk.

g, Cell viability of wild-type, Fancb, Neil3, or Fancb/Neil3 knockout CH12 cells after exposure to trioxsalen and UV-A irradiation. Two independent clones were used for the single mutants and three independent clones were used for the double mutant. Error bars, standard error of the mean. We speculate that, relative to HAP1 cells, CH12 cells may be more reliant on the FA pathway to repair trioxsalen-induced damage due to lower expression levels of NEIL3.