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 (Blömer et al., 1997; Guenzel et al., 2014). Vpr overexpression resulted in a marked increase in GFPnuc/RFPnuc ratios (Figure S6). Treatment of cells with the nuclear export inhibitor Leptomycin B resulted in a progressive increase in green fluorescence in the nucleus over time, paralleled by increasing GFPnuc/RFPnuc ratios, which were solely attributed to increasing mislocalization of 2G:NES over time (Figure S6). Most interestingly, fibroblasts derived from old donors showed relatively more GFP in the nucleus and more RFP in the cytoplasm (Figure 6C). Quantification revealed a significant impairment of NCC in old compared to young and middle-aged donor-derived cells (Figure 6D). Loss of NCC significantly correlated with donor age as well as with RanBP17 expression levels (Figures 6E and S6). Thus, using 2Gi2R as a sensitive NCC reporter, we were able to identify an age-dependent impairment of NCC in old donor-derived human fibroblasts. Further, as 3-week-old iNs showed aging transcriptome signatures, including decreased RanBP17 levels in the old, we asked
