Figure 3: Mutations of TRAF3IP1 impair IFT54 ciliary trafficking.

(a) Fibroblasts transfected with GFP–IFT54-WT were fixed with MeOH and stained for GFP (green), IFT54 (red) and acetylated α-tubulin (blue, cilia). The base and the tip of the cilia are indicated by arrowhead and asterisk respectively. Scale bar, 1 μm. (b) Traf3ip1-KD mIMCD3 cells stably expressing GFP-tagged IFT54 WT were fixed with 4% PFA and stained for Cep164 (distal appendages, red). Scale bar, 1 μm. (c) Ciliary distribution of IFT54 in serum-starved control and patients' fibroblasts stained for IFT54 (red), acetylated α-tubulin (green, cilia) and the basal body marker γ-tubulin (blue). Scale bar, 1 μm. (d) Percentage of cilia with IFT54 at the distal tip of cilia (arrows in (c), mean ± s.e.m. of n=4 experiments (that is, ≈100 cilia), ***P<0.001, Bonferronni's multiple-comparison test). (e) Distribution of IFT54 at the basal body in ciliated fibroblasts stained for IFT54 (red) and for γ-tubulin (blue) and centrin (green), markers of proximal and distal parts of centrioles, respectively. A schematic representation of the orientation of the two centrioles, with the localization of the distal (DAP) and subdistal (sDAP) appendages is shown. Scale bar, 1 μm. (f) Intensity of IFT54 staining at the transition fibres/transition zone (TZ, arrows in (e), mean ± s.e.m. of n=3 experiments (that is, ≈50 cilia), ***P<0.001, Dunn's post-hoc test). (g) Fibroblasts from control or affected individuals were fixed and stained for ODF2 (red, subdistal appendages) and IFT54 (green) and analysed by STED microscopy. A schematic representation of the orientation of the analysed centrioles is shown. Arrows in g indicate the pool of IFT54 present at the distal tip of the mother centriole corresponding to the transition fibers/transition zone. Scale bars, 0.25 μm.