Fig. 3

Cancer cell-intrinsic IL-15 activates the AKT-mTORC1-Cdc42 axis, leading to an increase in filopodium formation. (A), Phosphorylation of the indicated proteins was assessed by immunoblot analysis in A549 and PC9 cells transfected with siRNA-IL15 or siRNA-Ctrl or transduced with Lv-hIL-15 or Lv-hCtrl. (B and C), IL-15-overexpressing A549 and PC9 cells were treated with the PI3K inhibitor LY294002 (5 µM) (B) or the mTORC1 inhibitor rapamycin (500 nM) (C) for 24 h. Transwell migration assays were performed. Scale bar, 100 μm. The data in the bar graphs are presented as the means ± SDs and were analyzed with one-way ANOVA. ****, p < 0.0001. (D), A Cdc42 pulldown assay was performed on IL-15-overexpressing A549 and PC9 cells. (E and F), A549 and PC9 cells were transfected with siRNA-IL15 or siRNA-Ctrl for 48 h; subsequently, the cells were fixed and stained with Alexa Fluor 647-conjugated phalloidin to visualize F-actin (E). The same assay was performed on A549 and PC9 cells transduced with Lv-hIL-15 and Lv-hCtrl (F). The indicated number of cells (n) was used to quantify filopodia per cell. Two independent experiments were performed. Scale bar, 10 μm. **, p < 0.01; ****, p < 0.0001. (G-I), IL-15-overexpressing A549 and PC9 cells were cocultured separately with the inhibitors LY294002 (5 µM) and rapamycin (500 nM). The activity of Cdc42 was assessed by immunoblot analysis (G). The cells were fixed and stained with phalloidin (H and I). Scale bar, 10 μm. ***, p < 0.001; ****, p < 0.0001