FIGURE 4.

HA secretion and surface coat formation is influenced by Rab10. A, control and Rab10 siRNA-treated MCF7-EGFP-HAS3 cells were induced with 0.25 μg/ml doxycycline for 24 h for EGFP-HAS3 expression, and the media were assayed for HA synthesis. Rab10 silencing significantly increased HA synthesis. B, MCF7-EGFP-HAS3 cells were transfected with EGFP-Rab10 and EGFP only (mock), and induced with 0.25 μg/ml doxycycline for 24 h, and the media were analyzed for HA. HA secretion was significantly decreased when EGFP-Rab10 was overexpressed. C, parental MCF7 cells without HAS3 overexpression were treated with Rab10 siRNA along with the control groups, and the media were collected 48 h later. Rab10 silencing increased HA secretion compared with the control groups. Data in A–C represent mean ± S.E. (error bars) of 5 independent experiments. *, p < 0.05; **, p < 0.01; ns, not significant (paired t test). D, HA surface coat formation was analyzed in MCF7-EGFP-HAS3 cells with 0.5 μg/ml doxycycline induction. Quantitative image analysis was done with a ×20 objective. A significant increase in HA surface coat was observed with Rab10 silencing, compared with the control and control siRNA groups. Data represent mean ± S.E. of three independent experiments from >1500 cells/group in 15 random fields. *, p < 0.05; **, p < 0.01; ns, not significant (one-way ANOVA, Tukey's test). E, representative images taken with a ×40 objective show the increased HA coat by Rab10 silencing. Nuclei (blue) were stained with DRAQ5. Scale bar, 20 μm.