Fig. 1
ADAMTS9 and ADAMTS20 localize to the cilium base. a–c Immunostaining for primary cilia (acetylated α-tubulin, green), and human or mouse ADAMTS9 (red), shows ADAMTS9 localization at the primary cilium base in serum-starved RPE-1 cells (a), IMCD-3 cells (b), and human dermal fibroblasts (HDFs) (c). d Co-immunostaining of γ-tubulin (green) shows ADAMTS9 (red) around the basal body. e–g Focal ADAMTS20 staining (red) is present at the base of the primary cilium of NIH-3T3 cells (e), and HDFs (f), but not RPE-1 cells (g). h ADAMTS20 (red) is adjacent to the basal body (γ-tubulin, green) of NIH-3T3 cells. i 3D-projection of deconvolution super-resolution confocal microscopy (DSCM) of RPE-1 cells (imaged at ×1000 magnification) shows vesicle-like ADAMTS9 staining (red) at the primary cilium base (CP, presumed ciliary pocket, white arrowhead). j, k Representative DSCM image used for determining size and geographic distribution of ADAMTS9+ vesicles (j) and scatter plot (k) summarizing the analysis. l, m ADAMTS9+ vesicles (red) are located lateral and distal (above) to CEP170 (green), which defines the lateral-most boundary of sub-distal appendages (315.5 nm ± 41.9 nm SD). n ADAMTS9+ vesicles (red) are located lateral to the distal appendages (CEP164 (green)) (311.2 nm ± 62.7 nm SD). The arrowhead shows apparent overlap that was nonoverlapping in 3D. Right-hand panels depict relative localizations and radial distances measured by DSCM. o Immuno-EM of ADAMTS9 in RPE-1 cells shows 5 nm gold localization (arrowheads) in a barrel-shaped zone (pink shading) surrounding the basal body and 625 nm ± 110 nm from its center. Secondary antibody-only negative control lacks gold particles. p, q High magnification images show 5 nm gold particles (red arrows) localized to the left (top image) or right side (bottom image) of the basal body in the ADAMTS9-stained TEM samples, whereas none are visible in the negative control. Yellow arrows point to the basal body and the sub-distal appendages. Scale bars = 500 nm in i, 2 μm in l–n and 200 nm in o–p
