NBN variant MMC drug sensitivity screen. (A) WT NBN or 25 NBN variants were expressed in NBN^−/− HEK293T LP cells. Successfully recombined cells were identified as mCherry⁺/BFP⁻ population, separated by flow cytometry, and cultured in media supplemented with MMC 20 nM for 10 or 14 days. NBN^−/− HEK293T LP cells expressing WT NBN or WT-like variants (green) became more tolerant to MMC-induced DNA damage, which resulted in higher proliferation compared with cells expressing NBN variants with reduced (orange) or loss-of-function (red) activity. MMC drug sensitivity was determined by the fold change in variant frequency before and after MMC exposure and was quantified by Illumina MiSeq of the barcode sequence for each variant. (B) NBN variant abundance during 20 nM MMC treatment for 10 and 14 days was measured as the fold change to day 0 of the normalized barcode reads. MMC sensitivity was classified as high (<0.75), moderate (0.75-1) and WT-like (>1). Each dot represents an average fold change of 9 measurements, which derive from 3 barcodes assigned to each NBN variant and each condition performed as triplicates. (C) NBN variant protein stability was plotted against NBN variant MMC sensitivity. P values were estimated using the Pearson correlation test (R).