Extended Data Fig. 1: RO 10 μM reversibly blocks cells in G2-phase, without triggering DNA damage.
A: Gating strategy for FACS analyses. Cells gated for subsequent analysis are enclosed in polygons; A: live cells, B: single cells; G1/S/G2-M: cell cycle phases, SSC: side scatter; FSC: forward scatter; PE-Texas Red: Intensity of propidium iodide; APC: BrdU intensity (when BrdU labelling was performed). For some experiments, cell cycle analysis (rightmost panel) is presented as histograms (cell counts as ordinates). B-E: FACS analysis of human lymphoblasts (JEFF), of primary fibroblasts (MRC5), and of immortalized Chinese hamster fibroblasts (GMA32) treated with RO. The concentrations used and times of treatment are indicated, NT: Non-treated cells. Each experiment was done once. F: Experimental scheme for G-I. Exponentially growing (exp. gr.) cells were treated as indicated then released in normal medium (t0) and analysed by immunofluorescence (IF) with anti-γH2AX antibodies at indicated times. G: IF analysis of JEFF lymphoblasts. HU 1mM is used as positive control. Note the presence of mitotic cells (white arrows) in the 30 min panel. Scale bars: 20 µm. The percentage of nuclei with foci in each sample was determined by eye counting (histogram). H-I: GMA32 cells were analysed by FACS (H) or IF (I) as in F; white arrows and scale bars as in G. For quantification, pictures of 20 microscopic fields for each sample were assembled and the intensity of global red fluorescence was determined with Image Gauge (histogram). Experiments shown in G-I were carried out once.
