FIGURE 3.

Localization and functional characterization of PCNA NES in HeLa cells. A, effect of NES1 and NES2 fusion on EGFP localization using either EGFP-N1 (upper panels) or EGFP-C3 (lower panels) plasmids, allowing us to fuse NES sequence at the amino- or carboxyl-terminal part of EGFP, respectively. HeLa cells were transfected with pEGFP-N1, pEGFP-C3, pNES1-EGFP, pEGFP-NES1, pNES2-EFGP, or pEGFP-NES2, and EGFP was detected by confocal scanning microscopy (original magnification ×630, scale bar = 20 μm). B, effect of LMB on NES1-EGFP localization. HeLa cells transfected with pNES1-EGFP were treated with either ethanol solvent (−) or 20 ng/ml LMB for 6 h prior to fixation and permeabilization. HeLa cells transfected with EGFP-N1 were used as controls. Nuclei were stained with propidium iodide (original magnification ×400, scale bar = 20 μm, left panel). C, the percentage of cells showing either strong nuclear localization (white bars, N>C) or strong cytoplasmic localization (black bars, N<C) was determined by direct counting. Counting was performed on a minimum of 100 transfected cells per experiment. The data are the means ± S.E. of three independent experiments, **, p <0.001, ANOVA test. D, effect of leucine into alanine mutations within NES1 on the NES1-EGFP localization. HeLa cells were transfected with pEGFP-N1, pNES1-EGFP, or pMutNES1-EGFP, and EGFP was detected with confocal scanning microscopy. Nuclei were visualized with Hoechst stain (original magnification ×400, scale bar = 20 μm, left panel). E, the percentages of cells showing nuclear and cytoplasmic EGFP localization were determined as in C. The data are the means ± S.E. of three independent experiments, **, p <0.001, ANOVA test.