Figure 2. The GTPase Rab7 is necessary for Troglitazone-mediated prevention of HGF-induced cell surface-directed lysosome trafficking, cathepsin B secretion, and invasion.

A) I.F. microscopy was performed on DU145 prostate tumor cells expressing either a non-target (scrambled) shRNA or a Rab7-directed shRNA to visualize lysosomes (red), actin (green), and nuclei (blue). Rab7 shRNA expression prevents Troglitazone-induced juxtanuclear lysosome clustering and causes lysosomes to traffic to the cell surface independent of HGF. B) Quantitation of the spatial distribution of lysosomes is shown as mean distance from individual cell nuclei for each treatment condition. Error bars represent the s.e.m of 30 cells from at least three independent experiments. C) NT and Rab7 shRNA expressing DU145 cells were treated for 24 hrs with Troglitazone and/or HGF and the amount of Cathepsin B secreted into the culture media was detected utilizing a cathepsin B activity assay (see methods and materials). Cathepsin B secretion is increased and Troglitazone no longer prevents secretion in the Rab7 knockdown cells. Inset indicates LAMP-1 and Rab7 expression in the nontarget (NT) or Rab7 shRNA (KD) stable cell lines by Western blot. D) NT and Rab7 shRNA expressing DU145 cells were seeded onto Matrigel-coated transwell inserts and allowed to invade for 24 hrs. Treatments indicated were added to both the top and bottom of the insert. *Statistical significance (p<0.001) versus NT control; **Statistical significance (p<0.01) versus NT control. Scale bars: 10 µm.