FIGURE 6.

Retrograde traffic of Dendra2-HAS3 from cell surface is decreased by Rab10 silencing. A, siRNA-treated MCF7 cells were transfected with Dendra2-HAS3. The ROI was set to the cell surface using the inner edge of the HA coat, visualized by fHABC, as a marker. The selected region (white line) was photoconverted with 15% of the 405-nm laser power for 1 s. In control siRNA cells (top set of panels), the photoconverted Dendra2-HAS3 (red channel) from the cell surface was seen inside the cytoplasm already in 2–6 min, whereas the retrograde traffic of Dendra2-HAS3 (red channel) was slow following Rab10 silencing (bottom set of panels), even after 16 min. Scale bar, 20 μm. B, the kinetics of the Dendra2-HAS3 photoconverted to red in the cell surface was measured for a time span of 16 min at 2-min intervals. Rab10 silencing decreased the traffic of HAS3 from the cell surface to other parts of the cell. In the Rab10 silencing group at 16 min, on the average, 73% of the photoconverted Dendra2-HAS3 was left in the cell surface, whereas only 31 and 30% of the photoconverted Dendra2-HAS3 remained there in the control and control siRNA groups, respectively. Data represent the mean ± S.E. (error bars) of 3–5 independent experiments with 10 images per treatment and experiment. **, p < 0.01; ***, p < 0.001 for control versus Rab10 siRNA and control siRNA versus Rab10 siRNA, respectively (one-way ANOVA, Tukey's test).