Ciliary TRPP2 immunolabeling is not affected by knocking out TRPM3.

(A) For each of the wild-type (WT), TRPP2-knockout (TRPP2 KO), and TRPM3-knockout (TRPM3 KO) cell lines, representative maximum intensity projections through a Z stack of images are shown. The cells were immunolabeled for ARL13B, a ciliary protein [34] (first column). The ARL13B labeling was used to define ciliary volumes in the 3D data set as described in Materials and Methods. The maximum intensity projection for the TRPP2 labeling in those ciliary volumes (second column) shows that knocking out TRPM3 does not alter the TRPP2 ciliary labeling. Knocking out TRPP2 reduces the TRPP2 ciliary fluorescence but non-specific labeling remains. Bar = 10 μm. All images had contrast enhanced in the same manner. A few brightly labeled pixels were saturated so that lightly labeled pixels would be visible while keeping gamma at 1. No pixels were saturated for the quantification of intensity used to make the graph in (B). (B) A box plot of the median intensity of TRPP2 immunolabeling/ciliary volume for each cell line. Knocking out TRPM3 does not alter the TRPP2 ciliary labeling (P = 0.62 and P ≥ 0.99, Kruskal-Wallis one-way ANOVA on ranks with Dunn’s all pairwise comparison). Knocking out TRPP2 reduces the TRPP2 ciliary fluorescence by about half compared to the other lines (* P 19]). The box plot's top, middle, and bottom lines indicate 75th, 50th, and 25th percentile values, respectively. Whiskers indicate 90% and 10% values; circles indicate the 95% and 5% values. The numbers below each box plot indicate the number of ciliary volumes followed. Four cell passages were used for each cell line.