Validation by ciliary localization.

A) Fluorescence microscopy of eCFP fused to C20orf26, CCDC147 and IQCA1 in hTERT-RPE1 cells. Acetylated alpha tubulin (red) is used to mark the axoneme. DAPI (blue) staining is used to mark the cell nucleus. IQCA1 does not appear to co-localize with acetylated alpha tubulin. B) Localization of CCDC113, RIBC2, ARMC3 and C6orf165 (red) compared with acetylated alpha tubulin (green) in human lung epithelial cells. C) Localization of CCDC113 in the primary sensory cilium of mature mouse photoreceptor cells. On the left: indirect 2-color immunofluorescence of CCDC113 (green) and centrin-3 (Cen3, red), a marker protein for the connecting cilium (CC), the basal body (BB) and the adjacent centriole (Ce), and of the ciliary rootlet (CR) marker rootletin (green) and Cen3 (red) indicates the localization of CCDC113 at the ciliary base and the CR projecting into the inner segment (IS). On the right: immunoelectron microscopy of CCDC113 confirms the localization of CCDC113 at the CR (arrowheads) and demonstrates accumulation of CCDC113 in the periciliary region of photoreceptor cells (asterisks). D) On the left: localization of C6orf165 in the primary sensory cilium of mature mouse cone photoreceptor cells. Indirect double immunofluorescence of C6orf165 (green) and Cen3 (magenta) in combination with the counterstaining with fluorescent PNA (red) revealed the localization of C6orf165 at the BB and the Ce of cone photoreceptors and a punctate staining in the IS (arrowheads). At the center: higher magnification of the double immunofluorescence of C6orf165 (green) and Cen3 (red). On the right: immunoelectron microscopy of the ciliary region of photoreceptors confirmed the ciliary and periciliary localization (asterisks) of C6orf165, but also demonstrated its presence in outer segments (OS) of cones. E) Double immunofluorescence of RIBC2 (green) and Cen3 (red) of a photoreceptor cilium showed localization of RIBC2 throughout the connecting cilium (CC) and the adjacent centriole (Ce) as seen by co-localization with the ciliary marker Cen3. F) Double immunofluorescence of ARMC3 (green) and Cen3 (red) of a photoreceptor cilium revealed the absence of ARMC3 from the CC (counterstained for Cen3) but a punctate staining in the periciliary region of the photoreceptor IS (arrowheads). G) Localization of CCDC113, RIBC2, ARM3 and C6orf165 compared with polyglutamylated tubulin in hTERT-RPE1 cells. H) Positive predictive value (PPV) of the Bayesian classifier based on the experimental validation outcomes plotted against CiliaCarta gene rank. The PPV of the combined validation converges to 0.67, which equals the predicted PPV (0.67, given 0.33 FDR). The asterisks (*) above the x-axis denote the ranks of the candidate genes and proteins tested for ciliary function or localization.