Figure 6
From: Initial activation of EpCAM cleavage viacell-to-cell contact
EpICD nuclear translocation is mandatory for function. (A) HEK293 cells were stably transfected with ERT or EpICD-ERT expression plasmids. Expression of ERT and EpICD-ERT was assessed upon immunoblotting with ER- and EpICD-specific antibodies in combination with HRP-conjugated secondary antibody. Shown are representative results from three independent experiments. (B) HEK293 EpICD-ERT were treated with 4-hydroxytamoxifen (4-OHT, 100 nM) or diluent only (w/o 4-OHT) for 1 hr. EpICD was stained with specific antibodies (red) and nuclear DNA was visualised with DAPI (blue). Sections were recorded with a laser scanning confocal microscope. Shown are representative images from three independent experiments with multiple sections each. (C) HEK293ERT and HEK293EpICD-ERT cells were plated (3 × 105/plate) and cultured in the presence or absence of 4-OHT for three days. Cell numbers were assessed upon trypan blue counting and mean numbers with standard deviations from two independent experiments are given (* p < 0.05). (D) HEK293EpICD-ERT cells were treated with 4-hydroxytamoxifen (4-OHT, 100 nM) or diluent only (w/o 4-OHT) and c-Myc expression was assessed upon immunoblotting with specific antibodies and HRP-conjugated secondary antibodies. For a control, actin expression levels were assessed in parallel. n-fold induction of c-Myc was assessed upon densitometry and is given as the ratio of tamoxifen- versus control-treated cells. Shown are representative results from two independent experiments.