FIGURE 8:

Inhibitors of ANO1 channel activity have a negative effect on cilium length. (A) Quantification of the effect of ANO1 inhibitors on ciliary length in mpkCCD₁₄ cells. (B) Quantification of the effect of ANO1 inhibitors on ciliary length in IMCD3 cells. Solid squares, mean. Whiskers, 1 SEM. Box, 25th and 75th percentiles. Notch, median and 1–99% confidence intervals of the median. *p < 0.001 by two-tailed t test compared with the matched DMSO control. Each data point is the mean of 84–110 cilia measured in randomly selected fields. (C) Representative images of DMSO (control) and MONNA-treated IMCD3 cells labeled for F-actin (magenta) and expressing EGFP-tagged somatostatin receptor 3 (SSTR3-EGFP, green). In C, MONNA was added to the medium at the same time serum starvation was initiated. This protocol tested the effect of ANO1 inhibitors on cilium formation, elongation, and maintenance (labeled “elongation”). (D) Quantification of the effect of ANO1 inhibitors added for 6 h after 24 h of serum starvation. This protocol tested the potential effect of ANO1 inhibitors on maintenance of ciliary length (labeled “maintenance”). (E) Representative image of DMSO (control) and MONNA-treated IMCD3 cells labeled for F-actin (magenta) and expressing EGFP-tagged somatostatin receptor 3 (SSTR3-EGFP, green). Under both conditions of ANO1 inhibitor exposure (C, E) the somatostatin receptor continues to localize to the primary cilium. Vertical scale bars, 2.5 μm (C, E).