Extended Data Fig. 6: FAT1 loss results in chromosomal instability in lung cancer cells.
a, Transient FAT1 siRNA knockdown induces the formation of 53BP1 nuclear bodies in T2P cells. The boxes represent the interquartile range, the lines represent the median and the whiskers range denotes 1.5 x interquartile ranges, Dunn’s test, red bar = mean, n=3 biological replicates. Scale bar =10 µm. b, Transient FAT1 siRNA significantly increases the number of lagging chromosomes per cell after replication stress (left, centromeric; right, acentric), Dunn’s test. Over 100 mitotic cells were scored across 3 biological replicates. Red bar = mean. c, Representative metaphase spreads of FAT1 WT and KO A549 cells after 24 hours of aphidicolin treatment. Scale bar = 5µM d, Metaphase chromosome number following transient FAT1 knockdown in H1944 cells. A significant increase in metaphase chromosome number is observed in H1944 cells following FAT1 knockdown. N=3 biological repeats, red bar = mean. 2-sided Welch’s T-test. e, Mitotic error rate in PC9 cells following transient FAT1 knockdown in PC9 cells. one-way ANOVA, N=4 biological repeats, mean±SEM. f, Western blot showing FAT1 knockdown efficiency in PC9 cells. FANCD2 monoubiquitylation and γH2A.X, or expression level of E2F7 are not significantly affected by FAT1 ablation. g, FAT1 knockdown leads to more replication fork stalling in genome-doubled PC9 cells. >600 forks were counted across 3 biological repeats, 2-sided Paired T-test, scale bar =20 µM. h–j, FAT1 knockdown significantly reduces interphase FANCD2 foci (h, histogram) and mitotic FANCD2 foci (i and j, histogram and representative image, respectively) despite the increased rate of fork collapse. The boxes represent the interquartile range, black horiziontal bar represent the median and the whiskers range denotes 1.5 x interquartile ranges, red line= mean, N=3 biological replicates, >1200 interphase and >120 mitotic cells scored per condition, Dunn’s test.
