Figure 10.

Downregulation of mTOR elevates the number of cells that display GFP-WIPI-1 puncta. (A) Human U2OS cells stably expressing GFP-WIPI-1 were transfected with 20 nM control siRNA or siRNA targeting mTOR. 48 h post-transfection the cells were treated with control medium (CM), rapamycin (RM), wortmannin (WM) or rapamycin plus wortmannin (RM/WM) for 2 h, and a representative western blot analysis from 3 independent experiments confirmed mTOR down regulation. (B) Fluorescence images were automatically acquired and (C) analyzed, and results (600–800 cells per condition, n = 3) expressed as GFP-WIPI-1 puncta-positive cells (left panel) or GFP-WIPI-1 puncta per cell (right panel). P-values: * < 0.05, *** < 0.001. Scale bars 20 μM.