Figure 3.

CPZ blocks endocytosis in AQP2-MDCK cells: Polarized AQP2-MDCK cells were grown on filters for 4 days (A to C). Cells were preincubated with methyl-β-cyclodextrin (MBCD) (10 mM), a nonspecific endocytosis inhibitor, or CPZ (100 µM) for 15 min before the addition of Rhodamine-transferrin (Rho-Tf) (25 µg/mL) to the basolateral side of the cells. After incubation and fixation, Rho-Tf is scattered in vesicles throughout the cytoplasm in the untreated cells (A), whereas the Rho-Tf accumulates at the plasma membrane in cells treated with MBCD (B) or CPZ (C). Images were acquired using a Nikon A1R confocal microscope. These images are representative of three independent experiments. All images were taken at a central Z-axis plane that contained the nucleus of the cells (Bars = 10 µm). Quantification of the images was performed using Volocity software (D). The plasma membrane was identified by WGA-Alexa Fluor 647 staining. The intensity of Rho-Tf in the membrane and the cytoplasmic areas was analyzed by manually defining regions of interest. The fluorescence in the nucleus was used as a background level that was subtracted from all other values. At least 50 cells were analyzed in each of the three sets of images. The result is expressed as the % of total Rho-Tf fluorescence intensity at the membrane. The result is expressed as the mean ± SD, and data were analyzed using one-way ANOVA multicomparison (*p < 0.001, n = 3).