Extended Data Fig. 2: Cytogenetic analysis of cells treated with Aph, ARO or HU −/+ ATRi.
A, B: Metaphase plates from JEFF and MRC5 cells treated with Aph 600 nM for 16 h. Chromosomes were stained with Giemsa, arrowheads point to breaks. Two biologically independent experiments were done. C, D: Examples of chromosome breaks (white arrowheads) at FRA3B/FHIT or FRA16D/WWOX in JEFF cells (C) and at FRA1L/NEGR1 or FRA3L/LSAMP in MRC5 (D). Biologically independent experiments were done twice (C) or once (D). Left panels: FISH with probes specific to fragile genes (green) in association with probes specific to the centromere of corresponding chromosomes when available (red). Right panels: Chromosomes counter-stained with DAPI. Contrast was enhanced to better visualise the breaks. E: Total breaks and breaks at FRA3L and FRA1L were determined in normal MRC5 fibroblasts. Results are presented as in Fig. 1c. Biologically independent experiments were done twice (total breaks) or once (FRA3L and FRA1L), each experiment corresponding to analysis of 100 metaphases. Data are presented as mean values for total breaks. F-H: Impact of DRC signalling on chromosome stability in HU-treated JEFF lymphoblasts. F: A biologically independent duplicate of western blots shown in Fig. 1d was carried out and signals on digital images were quantified. The histogram shows the results of the two biologically independent experiments, HU values are presented relative to that in non-treated cells (NT). Data are presented as mean values. G: Example of a metaphase plate displaying mitotic catastrophe (24% of all metaphases) induced in cells treated with HU + ATRi (as in Fig. 1g). Metaphase plates were prepared and analysed as in A. The arrows point to multi-broken chromosomes. Biologically independent experiments were done twice. H: Examples of an atypical break at FRA3B, FISH and Giemsa staining as in C. Atypical breaks at FRA3B were commonly observed in HU+ATRi (50% of FRA3B breaks) but were not seen in Aph- or HU-treated cells (two biologically independent experiments). Scale bars: 20 µm on metaphase plates (A, B, G) and 0,5 µm on chromosomes (C, D, H).
