Effects of 5-FU on apoptosis (a), cell cycle (b), and Fas, p21 and AQP3 mRNA levels (c). Cells were incubated with increasing doses of 5-FU for 48 hours (a) Apoptosis was quantified by double staining with Annexin-V conjugated to FITC and propidium iodide, and analyzed with a FACScan flow cytometer. (b) The DNA content was measured using propidium iodide staining and fluorescence-activated cell sorting analysis. (c) Real-time RT-PCR for each gene was performed, after RNA isolation using GAPDH as the internal control. mRNA expression levels are presented as arbitrary units in relation to control values as the reference. Results are the mean ± SE of a representative experiment measured in triplicate, three independent experiments were performed.