Figure 2. CEP290 localizes throughout the length of the CC and in close proximity to the microtubule doublets.

(A and B) SIM and STORM images of representative cilia from adult retina, immunostained for transition fiber protein CEP164 and axonemal lumen protein Centrin (A) or CEP164 and CEP290 (B). Row average intensity plots are shown. (C) SIM image of a representative cross section through the CC with separated channels to the right. The white line depicts the position of the average intensity line plot. (D–F) SIM and STORM longitudinal images of representative cilia immunostained for CEP290 and acetylated α-tubulin (AcTub, E) or glycocalyx label WGA (F). Row average intensity plots are shown. For CEP290/WGA-labeled cilia, high- and low-magnification wide-field Tuba1 antibody staining (green) overlay is shown. (G) Dot plot with averages and standard deviations of the lengths of CEP290 and centrin staining in the CC for SIM and STORM. (H) Dot plot with averages and standard deviations of the radii of CEP290, AcTub, and centrin in the CC for SIM and STORM. Thirty-three percent of the maximum intensity value of each channel was used as the boundary criterion for the measurement of each cilium. Measurements were compared with Student’s 2-tailed t test and 1-way ANOVA and Tukey’s post hoc test, respectively. (I) A color-coded schematic of a CC. Scale bar: 200 nm. Dotted line indicates CC/OS border. Measurements were from 3 different animals and SIM images of 75 cilia for CEP290, 30 for AcTub and 45 for centrin, or STORM images of 42 cilia from 6 animals for CEP290, 16 cilia from 3 animals for AcTub and 52 cilia from 3 animals for centrin. AcTub, acetylated α-tubulin; WGA, wheat germ agglutinin; CC, connecting cilium.