FIGURE 10.

HA-dependent reduction of cell adhesion with Rab10 silencing. A, MCF7-EGFP-HAS3 cells with doxycycline-inducible EGFP-HAS3 expression showed a dose-dependent reduction of cell numbers after 48 h. Data represent means ± S.E. (error bars) of three independent experiments. B, Rab10 silencing decreased cell numbers in MCF7-EGFP-HAS3 cells induced to synthesize HA with 0.25 μg/ml doxycycline. C, Rab10 siRNA also reduced cell numbers in parental MCF7 cells. The data represent means ± S.E. of five and four independent experiments in B and C, respectively. D, 48 h post-transfection, Rab10 siRNA inhibits the adhesion of MCF7-EGFP-HAS3 cells, induced with 0.25 μg/ml doxycycline, whereas 5 transduction units/ml hyaluronidase for 6 h counteracts this by removing the HA coat, both in the cells with doxycycline-induced HA synthesis (Control siRNA) and those where HA synthesis was further enhanced by Rab10 siRNA. E, in a similar fashion, the MCF7 parental cells without enhanced HA synthesis were treated with Rab10 siRNA for 48 h and with 5 transduction units/ml of hyaluronidase for 6 h. There was a significant decrease in cell adhesion with Rab10 silencing but no effect by hyaluronidase in control or Rab10 siRNA groups. F, MCF7 parental cells were treated with exogenous, soluble HA, and cell adhesion to collagen I was analyzed as above. There was no significant decrease in adhesion, except in the cells treated with 500 μg/ml HA. The data represent means ± S.E. of three independent experiments in D, E, and F. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant (one-way ANOVA, Tukey's test).