Figure 2.

Effects of anosmin-1 in tumor cell motility. (A) Serum-starved cells were treated with either SFM (negative control), 10 nM recombinant anosmin-1, or FBS (positive control). The average moving distance (μm) of 20 random cells tracked over 20 h are shown. Error bars indicate s.e.m. from five independent experiments. The P values calculated by two-way ANOVA between the SFM and anosmin-1-treated groups in each cell line are 0.0175 (LN229), 0.0037 (A172), and 0.0399 (U87MG), where *P≤0.05 or **P≤0.01 is considered significant. (B) Effects of KAL1 knockdown on A172 cell motility. The P values obtained from three independent experiments are 0.0395 (for shRNA 673), 0.0253 (for shRNA 675), and 0.0015 (for shRNA 676) when compared with the nontargeting control shRNA. (C) As indicated, LN229 cells were pretreated with chemical inhibitors or specific antibodies for 30 min before addition of anosmin-1 (labeled A). Only anosmin-1 treatment alone or with nonspecific IgG resulted in a significant increase in motility. Error bars indicate s.e.m. from three independent experiments. (D) LN229, A172, and U87MG cells endogenously express anosmin-1, uPA, and FGFR1 proteins at variable levels. See Supplementary Table 3 for the mRNA levels of each gene. (E) KAL1-shRNAs significantly knocked down the endogenous anosmin-1 protein as assessed by two different anti-anosmin-1 (mouse or rabbit polyclonal) antibodies. (F) Knockdown efficacy of each shRNA is indicated as the percentage of the remaining KAL1 mRNA assessed by qRT-PCR, compared with control shRNA, which was significant (***P≤0.0001) in all three shRNAs. qRT-PCR was performed in triplicates, from four independent experiments. Error bars indicate the s.e.m.