Fig. 1

Expression and trafficking of SNAP-hAQP5 construct in NS-SV-AC cells. (A) WB analysis for AQP5 (using anti-AQP5 antibodies) and β-actin in five clones (1–5) or pool (used as positive control prior to limiting dilution cloning) of NS-SV-AC cells stably transfected with SNAP-hAQP5. STD: molecular weight standards. (B-C) Cells (clone 3 of NS-SV-AC SNAP-hAQP5) were pretreated for 24 h without or with 50µM indomethacin (-INDO: +INDO) prior to treatment for 8 h without (CTRL) or with 10 µM FK, 0.1 µM TH, or both (FK + TH), in 3 independent experiments. (B) Violin plots (with median and interquartile range of 25–75% percentile) of the membrane upper quartile (UpQ) intensity. Statistical significance evaluated using Kruskal-Wallis test with post-hoc Dunn’s tests is indicated as ****: p < 0.0001. In the absence of INDO pretreatment, the medians with IQR are: 1 with IQR 0.821–1.240, n = 203 cells for CTRL; 0.913 with IQR 0.755–1.180, n = 191 cells for FK; 1.027 with IQR 0.796–1.266, n = 229 cells for TH; 1.018 with IQR 0.780–1.265, n = 209 cells for FK + TH. In the presence of INDO pretreatment, the medians with IQR are: 1 with 0.789–1.414, n = 304 cells for CTRL; 1.347 with IQR 1.108–1.849, n = 414 for FK; 1.331 with IQR 1.066–1.816, n = 414 for TH; 1.331 with IQR 0.963–2.709, n = 410 for FK + TH. (C) Representative confocal images of immunofluorescent staining with anti-AQP5 antibodies (red) and DAPI (blue); yellow arrows indicate AQP5 localization close to the plasma membrane. Membrane segmentation images are also provided for cells pretreated with INDO. The specificity of the anti-AQP5 antibodies and the negative control performed in the absence of primary antibody are shown in Fig. S6