Figure 1.

Short-term CHX treatment blocks DNA replication. (A) Representative images of U2OS cells treated with 10 μg/ml CHX (36 μM) or 2 mM HU for the timepoints indicated. Cells were subsequently pulsed with 10 μM EdU for 20 minutes (in presence of inhibitors), followed by fixation and fluorescent labeling. (B) Quantification of (A) as the percentage of 100 cells counted in each experiment (n = 3). (C) Schematic representation of experimental setup of the fiber assay and representative images of DNA fibers. (D) Average replication speed of control or CHX (10 μg/ml) treated U2OS cells (n = 2). (E) U2OS cells were left untreated or treated with 0.4 μM aphidicolin (APH) for 24 hours. When indicated, cells were pre-treated with 10 μg/ml CHX or 2 mM HU 10 minutes prior to APH addition and kept in the media for the duration of the experiment. Cells were fixed and immunostained for 53BP1. Note that cells treated with HU and APH still induce 53BP1 foci (as seen in the image), whereas 53BP1 nuclear body formation is diminished. (F) Quantification of immunofluorescence in (E) as the percentage of 100 cells containing >10 53BP1 nuclear bodies (NBs) in each experiment (n = 2). The error bars depict standard deviation; *P ≤ 0.05, ** P < 0.01, ***P ≤ 0.001 as determined by Student's t-test. See also Figure S1.