Effect of FBXW 7 overexpression in glioma cells. Immunodetection of FLAG-Fbxw7 (green) in U87, 48 hours after transfection with plasmids encoding each of the three isoforms α (A), β (B), γ (C). Nuclei are labeled with DAPI (blue) and actin cytoskeleton with rhodamin-coupled phalloidin (red), bar = 50 μm. α-Fbxw7 localizes to the nucleus, β-Fbxw7 is cytoplasmic, γ-Fbxw7 localization varies. It is restricted to the nucleolus at low levels (insert) and leaks in the nucleus and in the cytoplasm at higher doses. Example of PCNA detection (red) in U87 cells overexpressing FLAG-Fbxw7 (green) α (D), β (E), γ (F). In lower panels (G-I), only the red signal in the same fields is shown. An example of α-Fbxw7-positive PCNA-negative cell is shown with arrowhead. bar = 10 μm. Quantification analysis of PCNA expression 48 h after transfection (J). Scoring was established on >150 cells per assay. The results (mean of two independent experiments, ± SEM) are expressed as the ratio of PCNA+ cells in FLAG+ versus FLAG- cells from the same transfection well. Quantification analysis of Ki-67 expressing cells in FLAG- and FLAG+ cells 48 h after transfection (K). Statistical analysis was performed using the Fisher's exact test (n > 150 cells for each group). The experiment was repeated twice with similar results. Cell growth after transfection with each isoform expression plasmid (L). For each assay, cells were stained with DAPI and anti-FLAG antibody 24 h and 72 h after transfection. FLAG positive and negative cells were scored from 20 independent 40×-magnified fields and their 72 H/24 h ratio compared. Cells overexpressing nuclear isoforms α and γ are significantly counterselected. Statistical analysis was performed using the Fisher's exact test (n > 1000 cells for each assay). The experiment was repeated twice and analyzed once after 48 h with similar results.