Figure 2

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.