Expression of IFT20 is down-regulated following suppressed expression of Ror2 in SaOS2 cells. (a) Quantitative RT-PCR analysis showing decreased expression levels of IFT20 in si-Ror2-transfected SaOS2 cells. Data are expressed as mean ± SD of four independent experiments (**P < 0.005, t test). (b) Western blot analysis showing decreased protein levels of IFT20 in SaOS2 cells transfected with either Ror2 or IFT20 siRNA. Whole cell lysates from the respective cells were analyzed by Western blotting with antibodies against the indicated proteins. The histograms indicate the relative levels of IFT20 and Ror2. Data are expressed as mean ± SD of four independent experiments (**P < 0.005, t test).

IFT20 plays important roles in invadopodia formation. (a) Suppressed expression of Ror2 or IFT20 inhibits invasive migration of SaOS2 cells. SaOS2 cells were transfected with the indicated siRNAs and analyzed by Transwell invasion assay. Cells invaded to the lower surface of the Transwell membranes were counted. Data are expressed as mean ± SD of three independent experiments (**P < 0.01, t test). (b) SaOS2 cells transfected with the indicated siRNAs were cultured on glass coverslips pre-coated with fluorescein-conjugated gelatin (FL-gelatin) for 6 hr and stained with rhodamine-conjugated phalloidin for F-actin. Insets show magnified images of boxed regions. Note that suppressed expression of Ror2 or IFT20 inhibits dot-like accumulation of F-actin and degradation of the FL-gelatin, signs of invadopodia formation. Scale bar, 10 µm. (c) Quantification of the data shown in (b). Number of invadopodia, identified as F-actin dots in the areas of degraded FL-gelatin, per cell was counted. Data are presented as a box-and-whisker plot. n = 127–178, three independent experiments; ***P < 0.001, t test. (d,e) Reduced activity of invadopodia formation 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-resistant (sr)-IFT20 (+) or pIRES2-ZsGreen1 (−), as indicated. The respective transfected cells were subjected to Western blotting with antibodies against the indicated proteins (d) or plated on glass coverslips pre-coated with Alexa Fluor 596-conjugated gelatin (Alexa-gelatin) (e). Invadopodia formation of ZsGreen1-positive cells were assessed and quantified as in (c). Data are presented as a box-and-whisker plot. n = 40–44, three independent experiments; **P < 0.01, t test.

IFT20 is required for reorientation of the centrosome toward the direction of cell invasion. (a) Localization of IFT20 at the cis-Golgi in SaOS2 cells. Cells transfected with si-Ctrl or si-IFT20#1 were cultured on FL-gelatin-coated glass coverslips for 6 hr. Cells were then stained with antibodies against IFT20 and cortactin, and counterstained with an antibody, which is conjugated to Alexa Fluor 647 and directed against GM130, a cis-Golgi marker. Boxed regions are magnified on the middle of each image. Note that invadopodia, indicated by a dot-like accumulation of cortactin in the areas of degraded FL-gelatin, are formed in the close vicinity to the Golgi apparatus, where IFT20 is localized. Scale bar, 10 µm. (b) SaOS2 cells transfected with the indicated siRNAs were examined for reorientation of the centrosome using 2D invasion assay (see Methods). The percentages of the edge cells in which the centrosome were detected within the 120° sector emerging from the center of the nucleus and facing toward the space were measured. The red line in the graph indicates the level of expected random orientation of 33%. Data are expressed as mean ± SD of three independent experiments (**P < 0.01, ***P < 0.001, t test).

IFT20 is required for assembly of the Golgi apparatus. (a) SaOS2 cells transfected with the indicated siRNAs were stained with antibodies against GM130 and γ-tubulin to visualize the cis-Golgi and centrosome (centriole pair), respectively, and counterstained with DAPI (blue). Insets show magnified images of boxed regions. Scale bar, 10 µm. (b) Number of Golgi fragments per cell was quantified and presented as a box-and-whisker plot. n = 69–74, three independent experiments; ***P < 0.001, t test. (c) Golgi dispersion in Ror2- or IFT20-knockdown cells can be suppressed by ectopic expression of IFT20. SaOS2 cells transfected with si-Ctrl, si-Ror2 or si-IFT20#1 were further transfected with pIRES2-ZsGreen1-sr-IFT20 (+) or pIRES2-ZsGreen1 (−), as indicated. Number of Golgi fragments in ZsGreen1-positive cells was quantified and presented as a box-and-whisker plot. n = 52–70, three independent experiments; *P < 0.05, ***P < 0.001, t test. (d) Effects of suppressed expression of Ror2 or IFT20 in SaOS2 cells on distribution of the cis-Golgi and TGN marker proteins. SaOS2 cells transfected with the indicated siRNAs were stained with antibodies against GM130 and Golgin-97 to visualize the cis-Golgi and TGN, respectively, and counterstained with DAPI (blue). Insets show magnified images of boxed regions. Scale bar, 10 µm.

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.

IFT20 is associated with AKAP450 and GM130 and required to maintain expression of AKAP450 at the cis-Golgi. (a) Co-immunoprecipitation of AKAP450 and GM130 with IFT20 in SaOS2 cells. Whole-cell lysates (WCL) from SaOS2 cells were immunoprecipitated with anti-IFT20 antibody or Ctrl IgG. Immunoprecipitates (IP) and WCL were analyzed by Western blotting with antibodies against the indicated proteins. (b) SaOS2 cells transfected with si-Ctrl, si-Ror2 or si-IFT20#1 were stained with antibodies against GM130 and AKAP450. Note that siRNA against Ror2 or IFT20 inhibits the colocalization of GM130 and AKAP450. The asterisks indicate AKAP450 at the centrosome. Scale bar, 10 µm. (c) 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. Colocalization of GM130 and AKAP450 was assessed as described in (b) and quantified. Data are presented as a box-and-whisker plot. n = 31–43, three independent experiments; **P < 0.001, t test).

IFT20 is required for efficient anterograde transport through the Golgi. (a,b) cis-Golgi-to-TGN transport of VSVG is delayed by suppressed expression of IFT20 or Ror2. SaOS2 cells treated with either IFT20 (a) or Ror2 (b) siRNA were further transfected with expression plasmid for VSVG-Myc with or without expression plasmid for sr-IFT20 (a) or IFT20 (b). Cells were incubated at 15 °C to accumulate VSVG-Myc at the pre-Golgi and then shifted to 32 °C. At the indicated time points, colocalization of immunostained VSVG and TGN46, a TGN marker, was examined by confocal microscopy and quantified. Data are expressed as mean ± SEM of three independent experiments. n ≥ 15 in each time point; *p < 0.05; **p < 0.01; Mann Whitney test. Note that delayed transport of VSVG in IFT20- (a) or Ror2-knockdown cells (b) is rescued by ectopic expression of sr-IFT20 or IFT20, respectively. (c) IFT20 knockdown fails to affect kinetics of retrograde COPI transport from the Golgi to the ER. SaOS2 cells were transfected with either Ctrl or IFT20 siRNA along with expression plasmid for VSVG-KDELR-Myc. Cells were incubated at 32 °C and then shifted to 40 °C to allow Golgi-to-ER transport of VSVG-KDELR-Myc. At the indicated time points, colocalization of immunostained VSVG-KDELR-Myc and Giantin, a cis-Golgi marker, was examined by confocal microscopy and quantified. Data are expressed as mean ± SEM of three independent experiments. n ≥ 15 in each time point. (d) Anterograde transport of VSVG-MT1-MMP through the Golgi is delayed by suppressed expression of IFT20. SaOS2 cells were transfected with either Ctrl or IFT20 siRNA along with expression plasmid for VSVG-MT1-MMP. Anterograde transport of VSVG-MT1-MMP was assessed as described in (a). Data are expressed as mean ± SEM of three independent experiments. n ≥ 15 in each time point; *p < 0.05; **p < 0.005; Mann Whitney test.