Tumor cell-specific nuclear accumulation of VP3. (A) GFP-VP3 fusion proteins expressed from transfection constructs used in this study. (B) CLSM images of SAOS-2 and SR40 cells 16 h after transfection (with 1 μg of plasmid DNA per 6 × 10⁵ cells and the Mirus TransitIT-LT1 transfection agent) to express the indicated GFP fusion constructs. (C) Quantitative analysis of the levels of nuclear accumulation (Fn/c after subtraction of background fluorescence), as determined by image analysis with the Image J public-domain software as previously described (5), from CLSM images such as those in panel B for SAOS-2 and SR40 cells and COS-7 and CV-1 cells. Results are the mean ± the standard error of the mean [n = ≥43 for SAOS-2 and SR40 cells and n = ≥20 for COS-7 and CV-1 cells for all constructs except GFP-VP3(1-73), where n = ≥7 and n = ≥15 for SAOS-2 and SR40 cells and COS-7 and CV-1 cells, respectively]. Statistically significant differences (Student's t test) between transformed and nontransformed isogenic cells are indicated. (D) Analysis of the levels of nuclear accumulation for GFP-VP3(1-121) (left) and GFP-VP3(74-121) (right) in low (Fn = <20)-, medium (Fn = 20 to 40)-, and high (Fn = >40)-expression SAOS-2 and SR40 cells (data from panel C). Results are the mean ± the standard error of the mean. NES, nuclear export signal.