Based on these data, we reasoned that the decrease in RanBP17 might indicate a possible functional impairment of the nuclear pore in the aged. As the most prominent function of the nuclear pore is to maintain proper nucleocytoplasmic compartmentalization (NCC), we established a reporter assay to measure NCC using lentiviral delivery of a dual reporter system (2Gi2R). This reporter consists of a fused double-GFP containing an NES sequence (2G:NES) and an internal ribosome entry site (IRES)-linked double-RFP containing an NLS sequence (2R:NLS) (Figure 6A). Increased false localization of green fluorescence in the nucleus along with a simultaneous loss of nuclear red fluorescence (GFPnuc/RFPnuc), thus represents a measure for loss of NCC (Figure 6A). To test whether there is a detectable difference in NCC between cell populations, we measured GFPnuc/RFPnuc in nuclei of fibroblasts using automated region of interest (ROI) selection based on binarized DAPI images from confocal sections (Figure 6B). To first test the applicability of our NCC assay, we overexpressed the HIV-1 protein Vpr in young fibroblasts since Vpr is suspected of altering the integrity of the nuclear envelope
