Analysis of the nuclear/cytoplasmic compartmentalization of Gal4-KRAB fusion proteins containing a strong nuclear export sequence (NES).

Cells were transfected with the indicated Gal4 fusion proteins, fixed and stained for Gal4 and (in human cells) for TRIM28. Cells with Gal4 expression were manually scored under the fluorescence microscope to contain excess nuclear (Nuc>Cyt), excess cytoplasmic (NucA: Example fluorescence images from human HeLa cells (corresponding channels for Gal4 and TRIM28 staining of the same image pane one below the other). Arrowheads mark some of the foci with the telltale simultaneous Gal4-KRAB and TRIM28 accumulation. Bar = 10 µm. B: Quantification for human HeLa cells (7–9 independent experiments for each construct; between 134 and 442 cells counted per experiment) 24 hours post-transfection and Xenopus laevis A6 cells (4–7 independent experiments, between 154 and 447 cells counted) 48 hours after transfection. Two-sided T-tests looking at the data between ZNF10-KRAB-AB and XFIN-KRAB-AB Gal 4 fusions indicated p-values of p = 1.7×10−6 (HeLa) and p = 0.23 (A6), respectively. Exact numbers of scored cells and complete statistical analysis can be found in Table S3.