Figure 4.

5-FU induces FOXO1 expression in a HuR-dependent manner. MDA-MB-231 cells were seeded into 6-well plates and treated with 5 μg/ml 5-FU. At the indicated time, cells were collected and (A) RT-PCR and (B) western blot analysis were conducted to detect HuR and FOXO1 expression. (C) MDA-MB-231 cells were seeded into 6-well plates and transfected with siRNA-Con, siRNA-HuR and FOXO1 overexpression and control lentivirus, respectively. After 24 h, cells were treated with 5-FU. Following 24 h of incubation, the apoptotic cells were measured by PI and Annexin V-FITC staining and analyzed by flow cytometry. Values are expressed as mean ± SEM of at least three independent experiments. ^*P<0.05. (D) Cells were treated as described above. Caspase-3 activities were measured using a Caspase-Glo 3/7 assay kit. Values are expressed as mean ± SEM of at least three independent experiments. ^*Indicates P<0.05. 5-FU, 5-fluorouracil; HuR, human ELAV/Hu protein; FOXO1, Forkhead box protein O 1; Con, control. RT-PCR, reverse transcription-PCR; PI, propidium iodide; FITC, fluorescein isothiocyanate.