FIG. 4.

(A) Tim prevents PCC. Analysis of PCC by FACS. HeLa cells were transfected with control or Tim siRNA and then either mock treated or treated with HU (2 mM) for 20 h. Cells in mitosis were determined by staining with propidium iodide and antibody to P-H3, and the percentage of the P-H3 reactivity in S-phase cells by flow cytometry was considered as PCC. One representative of three experiments is shown. Data are expressed as percentages of the control samples (control siRNA) and plotted as the means ± standard deviations. Quantitation of the data represents the averages of three independent experiments. (B) Tim prevents PCC after replication stress. HeLa cells were transfected with control or Tim siRNA and then either treated with HU (2 mM) for 20 h or left untreated. To obtain M-phase-enriched cells, cells were additionally treated with Colcemid in the last 4 h. Mitotic spreads were prepared, and cells that had characteristic features of either a normal mitosis or PCC were determined by fluorescence microscopy. Interphase cells and cells that were intermediate in morphology between normal and PCC were not counted. The three frames on the left show three characteristic DNA-staining patterns. For quantitative analysis approximately 100 mitotic cells were counted per condition. The values represent the means of three independent experiments, and the error bars indicate standard deviations. (C) Tim inhibition causes RDS. HeLa cells transfected with control or Tim siRNA were grown in the presence of [¹⁴C]thymidine for 40 h to label DNA uniformly until the second transfection and then grown in nonradioactive medium for an additional 24 h. Cells were exposed to UV (2 J/m²) or left untreated, incubated at 37°C for 30 min, and then labeled for 15 min in medium containing [³H]thymidine. Relative DNA synthesis was estimated from the incorporated [³H]thymidine normalized to total DNA by the ¹⁴C radioactivity. Data are expressed as percentages of the control samples (no UV irradiation) and plotted as means ± standard deviations. The data represent the averages of three independent experiments.