Figure 2. The translation inhibitor gephyronic acid A strongly reduces P-body formation in HeLa cells. (A) Cells were treated with either 1 µM of gephyronic acid A (GA) for 3.5 h, 25 µg/ml of cycloheximide for 15 min, 100 µg/ml of puromycin for 1 h, or mock-treated. After the indicated time, cells were immunostained with an antibody for DDX6 (green) and the nuclei was visualized with DAPI (blue). The number of DDX6-containing P-bodies from at least 100 cells per condition was quantified and related to those detected in mock-treated cells. Representative images for each condition are shown. Numbers below the panel show the median and the standard error of the mean of the P-body number per cell relative to the untreated control (set to 100%). (B) HeLa cells were incubated for 1 h (upper graph) or 3.5 h (lower graph) with 0, 50, 100, 500, or 1000 nM of GA. Cells were immunostained to quantify DDX6- and Dcp1-containing P-bodies in at least 100 cells. The relative abundance of DDX6- and Dcp1-containing P-bodies vs. log GA concentration is shown related to mock-treated cells. Right graphics indicate cell viability for the different concentrations of GA and times of incubation using an ATP assay that measures intracellular ATP levels. Error bars indicate standard error of the mean. (C) For both time-points, expression levels of DDX6 and Dcp1 were tested by western blot under the higher concentrations of GA. β-actin was used as a control for protein loading.