Extended Data Fig. 7: FAT1 loss promotes WGD and multinucleation.
a, Proportion of FAT1 driver mutations in genome-doubled TRACERx421 tumors. Fisher’s Exact Test, p=0.179. b, FAT1 CRISPR KO leads to elevated MCM7 reloading rate >6N in TP53 mutant PC9 cells, 2-sided Paired T-test, mean±SD, n=3 biological replicates. c, FAT1 ablation leads to elevated MCM7 reloading rate >4N in TP53 WT RPE1 cells with transient FAT1 siRNA transfection, mean±SEM, 2-sided paired T-test, n=3 biological repeat. d,e, Cyclin B1 levels in the G2/M population following CTRL or FAT1 depletion in TP53 WT RPE1 cells. The histogram in d shows no difference in G2/M cyclin B1 levels, mean±SEM, 2-sided paired T-test, n=3 biological repeats. The representative dot plots in e show cyclin B1 levels of the G2 population. f, Nocodazole treatment arrests PC9 cells at mitosis and causes endoreplication (EdU positive cells >6N). FAT1 knockdown does not significantly further increase EdU-positive cells >6N when treated with nocodazole, mean±SEM, 2-sided paired T-Test, n=4 biological replicates. g, Live cell tracking data showing mitotic error rate of RPE1-TERT-FUCCI cells. 2 different FAT1 siRNAs produced a significant increase in mitotic error rate. One-way ANOVA with Sidak correction, mean±SD, n=3 biological repeats. h, Quantification of fixed microscopy images showing that FAT1 siRNA knockdown increases the rate of multinucleated U2OS cells, with or without replication stress induced with hydroxyurea. Left, multinucleation was quantified using phalloidin as a marker for cell boundaries and nuclear envelope stain emerin was used to mark the number of nuclei per cell. Mean±SD, 2-sided paired T-test, Biological repeats: n=4 without damage, n=6 with HU. Right, representative image of cells, scale bar = 10 μM, white arrows = multinucleated cells. i, Quantification of fixed microscopy images showing FAT1 knockdown elevates EdU incorporation rate in multinucleated TP53 KO RPE1-TERT cells, but not in the TP53 WT counterpart. Following aphidicolin-induced replication stress, FAT1 depletion increases EdU incorporation rate in both TP53 WT and TP53 KO cells, mean±SD, one-way ANOVA with Holm-Sidak correction. Biological repeats, no damage n=3; Ctrl siRNA with aphidicolin n=3, FAT1 siRNA with aphidicolin n=4.
