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.