Figure 5.

EPSTI1 regulates tumor cell properties. A: EPSTI1-transfected MCF-7 cells serve as a model for conditional expression of EPSTI1. RT-PCR (upper panel, left) and Western blotting (upper panel, right) of EPSTI1 mRNA and protein, respectively, as regulated by 100 ng/ml doxycycline (Dox. +/−) in a MCF-7–EPSTI1–Tet-off cell line. Note the on/off quality of the expression both at the mRNA and protein level within a time frame of three days. Immunoperoxidase staining (lower panel) showing the typical staining primarily in the nucleus and to a lesser extent in the cytoplasm of the expressing cells (right) as compared with doxycycline treated cells (left, Scale bar = 50 μm). B: EPSTI1 expression facilitates anchorage independent growth in soft agar and tumorsphere formation. Left: Bar diagram depicting the quantitative difference in colony forming ability of MCF-7–EPSTI1–Tet-off cells with and without doxycycline. Each bar represents the mean ± SD from triplicate wells (*P < 0.01). Right: Histogram showing the frequency of single cell–derived tumorspheres from MDA-MB 231 cells induced to express EPSTI1 by IFN-α in the presence of either EPSTI1-siRNA2 (EPSTI1-siRNA) or control-siRNA2 (control-siRNA). A significant difference in favor of EPSTI1 expressing tumorspheres was recorded in both experiments. Each bar represents the mean ± SEM of three experiments performed in duplicate (*P < 0.05). C: Stem cell PCR array of MCF-7–EPSTI1–Tet-off cells with and without EPSTI1 expression. Genes associated with embryonic lineage property including ACTC1, FOXA2, ISL1, with mesenchymal cell lineage property markers of COL1A1, and COL9A1, and with a critical Wnt-mediated effector, DVL1 show at least twofold up-regulation. Data are presented as relative expression of genes in EPSTI1-expressing cells, compared with cells without EPSTI1 expression.