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. Author manuscript; available in PMC: 2017 Oct 1.
Published in final edited form as: Nat Genet. 2017 Feb 13;49(4):537–549. doi: 10.1038/ng.3790

Figure 6. Increased spontaneous chromosome breakage and fragmentation of mitotic chromosomes in DONSON-depleted cells.

Figure 6

(a,b) Metaphases chromosomes from DONSON or control siRNA transfected HeLa cells were visualised by Giemsa staining and light microscopy. (a) Quantification of average numbers of chromatid gaps/breaks per metaphase (n=6; >50 metaphases per sample per experiment). (b) Representative images of normal chromosomes, chromosomes containing gaps/breaks, highly fragmented and pulverized chromosomes. Red arrows denote chromatid gaps/breaks; blue arrows indicate chromosomal exchanges. Scale bar; 10 μm. (c–g) Loss of the structure-specific nucleases MUS81 or XPF significantly reduces the spontaneous replication fork asymmetry and genome instability in DONSON-depleted cells. (c) Cells transfected with the indicated siRNAs were pulsed with CldU and IdU. Replication fork asymmetry was measured as in (Fig. 3e). The red lines denotes median ratios (n=3). (d) Co-depletion of MUS81 or XPF with DONSON reduces levels of spontaneous DNA damage. Extracts from cells transfected with the indicated siRNAs were subjected to SDS-PAGE and immunoblotting using the antibodies indicated. (e–f) Co-depletion of MUS81 (e) or XPF (f) reduces chromosomal aberrations in cells lacking DONSON. Quantification of the average number of chromatid gaps/breaks per metaphase in cells transfected with control, DONSON, MUS81 and/or XPF siRNA. At least 50 metaphases per experiment were counted (n=3). (g) Quantification of the average percentage of metaphases containing highly fragmented chromosomes or pulverized chromosomes in cells transfected with the indicated siRNAs. At least 50 metaphases per experiment were counted (n=3).