Fig. 4.
Analysis by imaging flow cytometry of the degree of nuclear localization of hPPIP5K2-GFP and hPPIP5K2NLS-3A-GFP. Live HEK293T cells, transfected with either a, hPPIP5K2-GFP (n = 632; 44236 ± 1711 total cellular fluorescence units) or b, hPPIP5K2NLS-3A-GFP (n = 1195, 48900 ± 1367 total cellular fluorescence units), and treated with the nuclear DRAQ5 stain, were analyzed for the degree of co-localization of the two spectral signals in every nuclear pixel (approx. 700 per cell), using imaging flow cytometry. The histograms depict the frequency of binned Similarity-Scores [28]; best-fit Gaussian plots are added. The horizontal arrows highlight the percentage of cells in which the expressed hPPIP5K2 construct was determined to have a significant nuclear localization (Similarity Score > 1.2; [35, 36]). The median value (M) of the Similarity Score for cells expressing hPPIP5K2NLS-3A-GFP is significantly lower than that for cells expressing WT enzyme (p < 0.001; Mann–Whitney Rank Sum Test)