Fig. 5.

Subcellular localization of the AKT1 variants.

Fluorescence microscopy of HeLa cells transfected with constructs driving the expression of different AKT1 variants fused to the mCherry fluorescent protein, in the presence of serum. Images were obtained with a Zeiss ApoTome microscope. This instrument allows the production of optical sections by using structured illumination. The left panels represent merged Z-stacks of typical cells (i.e. several images taken at different depths/focal planes within the cell). DNA was stained with Hoescht (in blue). The right panels represent optical sections, where DNA staining is not shown, to better appreciate the sub-cellular distribution of the AKT1 variants. Note the rather diffuse distribution of wild-type AKT1 and the membrane and the nuclear enrichment of E17K (activating mutation). The typical staining pattern of the mutated AKT1 carrying the duplications is strikingly different. Note the strong enrichment in the cortical sub-membrane regions. Moreover, the transfected cells displayed a profusion of filopodia-like processes (see insert). Similar results were obtained in the absence of serum. Further examples are provided in the Supplementary material.