Fig. 4

Basal body maturation is impaired in ADAMTS9-null cells. a Super-resolution imaging of distal (CEP164, red) and sub-distal appendages (CEP170, green) shows normal basal body formation in RB4 and D12 cells. b TEM of serum-starved wild-type and D12 cells shows D12 basal bodies (BB) with docked distal appendage vesicles (DAV) and ciliary vesicles (CV) (red arrows) but no axoneme. CP, ciliary pocket; DC, daughter centriole. c Quantification of basal body-associated vesicles at different stages of formation and growth shows that the majority of the mutant basal bodies do not progress beyond ciliary vesicle docking. N = 50 BB (WT), N = 100 BB (D12). d DSCM imaging of 24 h serum-starved wild-type and D12 cells stained for CP110 (red) and CEP170 (green) shows that the basal body of D12 cells retains the distal CP110 cap. e Quantification of basal bodies with and without CP110 caps (N = 3 independent experiments, ****p < 0.00001, Student’s t test). f Quantitative RT-PCR of CP110, EHD1 and EHD3 mRNAs in wild-type (Wt) and D12 RPE1 cells. N = 3 independent experiments; n.s., not significant; **p < 0.001, Student’s t test. g, h Western blot (g) and quantification (h) of CP110, EHD1 and EHD3 (red) normalized to GAPDH (green) shows higher CP110 in 24 h serum-starved ADAMTS9 mutant D12 cells but no change in EHD1/ EHD3 levels (N = 3 independent experiments, *p < 0.05, Student’s t test). i DSCM imaging of 6 h serum-starved wild-type RPE-1 cells co-stained with CP110 (red) and Rab11 (green) shows Rab11 + vesicles near CP110 cap of the basal bodies (arrowheads). j Staining for the transition zone (TZ) marker TCTN2 shows a TZ in wild type cells and the few mutant (D12) cells that produce ciliary axonemes (arrowheads). k Quantification of phospho-histone H3 positive cells show increased cell proliferation in non-serum-starved mutant cells and minimal proliferation upon serum starvation, similar to wild-type cells (N = 3 experiments, *p < 0.05, Student’s t test). l, m Schematic showing the ciliogenesis sequence³³ and arrest at the ciliary vesicle docking stage in the absence of ADAMTS9 (right). Scale bars = 500 nm in a–d, i. Bar charts and dotplots show mean and SD (error bars). In box and whisker plots, whiskers indicate the range and boxes mark upper and lower quartiles. The centerline indicates the mean