Fig. 4: FAT1 loss elevates replication stress and micronuclei.
a, FAT1 knockout exacerbates replication fork stalling in A549 cells. Top, scheme of the nucleotide labeling used to measure replication fork stalling. Bottom (left) quantification; (right), representative image for the DNA fibre experiments. The data represent means ± s.d. Statistical significance was determined by two-sided paired t-test (n = 3 biological replicates; >600 forks counted in total). Scale bars, 20 µm. b, TCGA LUAD analysis showing that FAT1 copy number loss is significantly correlated with weighted genome instability index measurements. The blue lines indicate FAT1 loss and the red dotted lines indicate the 90 and 95% confidence intervals. Confidence intervals were generated using computational permutation analyses. c, Box plot comparing the numbers of indels in FAT1 WT versus mutated tumours in the Genomics England LUAD and LUSC cohorts. The boxes represent interquartile ranges, the lines represent median values and the ranges of the whiskers denote 1.5× the interquartile range. Statistical significance was determined by two-sided Wilcoxon rank-sum test. n = 818 (WT) and n = 16 (mut). d, Transient FAT1 siRNA knockdown induces the formation of 53BP1 bodies in cyclin A-negative U2OS cells following 4 mM hydroxyurea for 5 h and recovery for 24 h. Statistical significance was determined by two-sided Wilcoxon rank-sum test. Scale bars, 10 μm. The red bars in the graph to the left represent mean values (n = 5 biological replicates). e,f, Transient FAT1 siRNA knockdown in U2OS cells induces the formation of total micronuclei with or without replication stress induced by 5 h of 4 mM hydroxyurea followed by 24 h recovery (e), as well as the formation of both acentric and centromeric micronuclei following the hydroxyurea treatment (f). The data represent means ± s.d. Statistical significance was determined by one-way ANOVA with Bonferroni correction. Biological repeats: n = 8 (e) and n = 4 (f). g,h, FAT1 loss elevates the rate of micronuclei formation in response to replication stress induced by 0.2 µM aphidicolin treatment (24 h) following FAT1 CRISPR knockout in A549 cells (g) or transient siRNA knockdown in T2P cells (h). The data represent means ± s.d. Statistical significance was determined by two-sided Student’s t-test. Biological repeats: n = 4 (total micronuclei in g), n = 3 (centromeric and acentric micronuclei in g) and n = 8 (h).
