Figure 4 DNA Catenanes Persist into Mitosis in Cells with TOP3A Mutations, Leading to Chromosome Segregation Defects and Genome Instability
(A and B) Chromosome segregation is impaired in TOP3A-deficient primary fibroblasts. (A) Representative images of chromatin bridges, lagging DNA (DAPI), and UFBs (detected by the presence of PICH and absence of DAPI stain). (B) Quantification of chromatin bridges, lagging DNA, and PICH-positive UFBs scored in control, parental, and P1 mitotic fibroblasts staged at anaphase B (experiments ≥ 3, n > 50 cells, error bars = SEM). To enrich for mitotic cells, we treated fibroblasts with R03306 for 6 hr and released and fixed them after 45 min.
(C) TOP3A P1 fibroblasts display significantly elevated amounts of micronuclei. Top: representative picture of control and P1 fibroblasts. Bottom: quantification of micronucleus containing interphase cells (experiments ≥ 3, n > 500, error bars = SEM). To enrich for G1 cells, we released RO3306-treated cells into fresh media for 30 min and collected, re-seeded, and fixed prometaphase cells after 4–6 hr.
(D) P1 fibroblasts with TOP3A mutations display significantly elevated numbers of 53BP1 bodies in G1 nuclei. Top: representative images of 53BP1 foci (red) and DNA (DAPI). Bottom: quantification of cells with at least four 53BP1 foci in G1 nuclei (negative for cyclin A) (experiments ≥ 3, n > 500, error bars = SEM). Scale bar: 2 microns. Two-tailed t test was performed against parent cells.