important functional signature of old human neurons. To confirm age-related NCC impairment, we next applied fluorescence loss in photobleaching (FLIP) as a second assay to measure compartmentalization to our aging fibroblasts. For this, we performed live-cell analysis of a shuttling 2xGFP:NLS:NES probe in young and old fibroblasts. FLIP revealed significantly longer half-life times in old donor-derived cells, indicating less efficient shuttling of the 2xGFP:NLS:NES probe (Figure S6). This observation represents an additional experimental measure supporting observed NCC defects as detected using the 2Gi2R assay.
