Fig. 5

MT aster formation is required for Cep290 localization at CS and TZ assembly. a–c Percentage of cells with CS Cep290 after treatment with DMSO, 20 μM nocodazole (a), 1 μM taxol (b), or 10 μM ciliobrevin D (c) for 30 min. d RPE-1 cells transfected with control or FSD1 siRNA were subjected to nocodazole treatment for 2 h and released at indicated time points. Next, cells were fixed with 4% paraformaldehyde in PBS and stained with antibodies to α-tubulin to visualize microtubules (green) and Centrin2 to mark centrosomes (red). Percentage of cells with different microtubule regrowth statuses at indicated time points was quantified on the right panel. Scale bars, 5 μm (main image) and 1 μm (magnified region). e RPE-1 cells were transfected with indicated siRNA were subjected to nocodazole treatment for 2 h and released at indicated time points. Cells were then fixed and permeabilized with cold methanol and stained with antibodies to Cep290 (red), α-tubulin (green), and Centrin2 (purple). Percentage of cells with CS Cep290 after nocodazole release for 20 min was quantified on the right panel. Scale bars, 5 μm (main image) and 1 μm (magnified region). f Effects of FSD1, Ninein, or Kif3a depletion on the Cep290 localization at centriolar satellites in cycling RPE-1 cells. g, h Effects of FSD1, Ninein, or Kif3a depletion on the relative intensity of the transition zone components TMEM67 (g) and NPHP8 (h) at mother centrioles in quiescent cells. Data are presented as mean ± s.d. of three independent experiments. n number of cells. In all panels, statistical comparisons between two groups were carried out by two-tailed t-test. NS not significant, **P < 0.01, ***P < 0.001