Effect of 5-fluorouracil on AQP3 expression (a) and cell volume (b) and effect of AQP3 silencing on cell growth inhibition induced by 5-fluorouracil (c). MCF7 cells were incubated for 90 minutes with 5-FU (FU) 250 μM and mRNA was isolated at 48 hours (a). Real-time RT-PCR analysis for AQP3 was performed using GAPDH as endogenous control. Data are calculated as arbitrary units relative to untreated cells (CT) as a reference. Results are the mean ± SE of three independent experiments measured in duplicate. At 48 hours after 90 min exposure to 250 μM 5-FU, MCF7 cells (b) were collected and volumes were measured as cell diameters (μm). Results are the mean ± SE of three independent experiments measured in triplicate. (c) Non-transfected (white), negative control siRNA (dashed) or AQP3-siRNA (black) transfected MCF7 were treated for 90 min with increasing doses of 5-FU (5 to 500 μM). After 48 h cells were trypsinized and the number of cells was measured using a cell counter. Cell viability is calculated as a percentage in relation to untreated cells as a reference. Results are the mean ± SE of three independent experiments measured in triplicate. Statistical significance was assessed with the Student’s t test (* p < 0.05; ** p < 0.01; *** p < 0.001).