Figure 4. FrAdV1 Pre-pVII contains multiple functional NLSs. (A) Schematic representation of the GFP-fusion proteins expressed, with NLSs represented as white boxes, along with their amino acid sequence and position. (B) HEK 293-A cells were seeded on glass coverslips and transfected to express the indicated GFP fusion proteins. 24 h post transfection cells were incubated with DRAQ5 to stain cell nuclei, fixed and coverslips mounted on slide holders before being analysed by using a Nikon A1 CLSM equipped with a 60x oil immersion objective. Representative images of the 633 nm (nuclei), 488 nm (GFP), and 568 (fibrillarin) laser channels are shown, along with a merged image (merge). Micrographs such as those shown were used for image analysis by measuring nucleolar (Fno), nuclear (Fn), cytoplasmic (Fc) and background (Fb) fluorescence for single cells using FiJi. (C) The levels of nuclear accumulation (Fn/c) were calculated using the formula Fn/c = (Fn-Fb)/(Fc-Fb). (D) The levels of nucleolar accumulation (Fno/n) were calculated using the formula Fno/n = (Fno-Fb)/(Fn-Fb). Data shown are individual measurements (circles ), along with means (black horizontal bars) + standard deviation of the mean relative to > 45 cells from three independent experiments per each GFP fusion protein, along with the results of Welch and Brown-Forsythe ANOVA statistical analysis as compared to GFP alone (/). **** = p <0.0001.