Figure 3.

Short-term cellular exposure to CHX is rapidly reversed whereas long-term exposure is toxic. (A) U2OS cells were treated with 10 μg/ml CHX or 2 mM HU for 1 hour, washed and released into fresh media for 1 or 5 hours, pulsed with 10 μM EdU for 20 minutes, followed by fixation and fluorescent labeling. (B) Quantification of relative fluorescence intensities in (A) (n = 2). Viability as determined by resazurin in U2OS cells (C) or immortalized BJ-hTERT cells (D) exposed to 10 μg/ml CHX or 2 mM HU for 24, 48 or 72 hours (n = 3). (E) Clonogenic survival assays of U2OS cells treated with 10 μg/ml CHX or 2 mM HU for 48 hours, followed by trypsinization and re-seeding of 500 cells in a 6-well format. After 10 days, cells were fixed and stained in 4% methylene blue-MeOH and quantified (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 S4.