IFT20 is required for MT nucleation at the Golgi. (a) Effect of suppressed expression of Ror2 or IFT20 on Golgi reassembly during MT repolymerization. SaOS2 cells transfected with the indicated siRNAs were treated with 0.5 µg/ml nocodazole (NZ) for 3 hr. After removal of NZ, cells were incubated for 0, 30, 60, and 90 min at 37 °C. Fixed cells were stained with anti-GM130 antibody (green) and DAPI (blue). Based on the pattern of the Golgi structures, cells were categorized into 3 classes as shown on the top panels: class 1 (cells with dispersed Golgi throughout the cytoplasm), class 2 (cells with dispersed Golgi around the nucleus), and class 3 (cells with normal compact Golgi). The mean percentages of cells in the respective classes were measured. n = 108–200, four independent experiments. (b) Effect of Ror2 or IFT20 knockdown on MT nucleation. SaOS2 cells transfected with Ctrl, Ror2 or IFT20 siRNA were treated with 3 µg/ml NZ for 2 hr. Cells were washed with ice-cold medium to remove NZ followed by 5 min incubation at 25 °C before fixation. Fixed cells were stained with antibodies against GM130 and tyrosinated (Tyr)-tubulin to visualize the cis-Golgi and newly nucleated MTs, respectively. Serial optical confocal z sections were obtained and stacked. Insets show magnified images of boxed regions. The arrows and arrowheads indicate Golgi-derived MTs (Golgi-MTs), one end of which is attached to a Golgi fragment, and non-centrosomal, non-Golgi-MTs, respectively. The asterisks indicate the centrosome. Inverted gray scale images of Tyr-tubulin were shown on the right. Scale bar, 10 µm. (c) Number of Golgi-MTs and non-centrosomal, non-Golgi-MTs per cell was quantified. Data are presented as a box-and-whisker plot. n = 50–56, three independent experiments; ***P < 0.001, N.S. = not significant, t test. (d) Reduced nucleation of Golgi-MTs by suppressed expression of Ror2 or IFT20 can be rescued by ectopic expression of IFT20. SaOS2 cells transfected with si-Ctrl, si-Ror2 or si-IFT20#1 were further transfected with pIRES2-ZsGreen1-siRNA-sr-IFT20 (+) or pIRES2-ZsGreen1 (−), as indicated. MT nucleation of the respective transfected cells was assessed as described in (c), and the number of Golgi-MTs in ZsGreen1-positive cells was quantified and presented as a box-and-whisker plot. n = 24–39, three independent experiments; ***P < 0.001, t test.