Figure 6: The cytoplasm-restricted expression of POH1 fails to increase E2F1-Survivin/FOXM1 expression.

(a) The nuclear (Nu) and cytoplasmic (Cy) fractions of tumour cells were analysed with antibodies against POH1, histone H2Ax (the indicative for nuclear fraction) and tubulin (a control for the cytoplasmic fraction). (b) The nuclear and cytoplasmic fractions of the SMMC-7721 cells stably expressing POH1-Flag were analysed using anti-Flag antibody. (c,d) Fluorescent staining of SMMC-7721 cells and the SMMC-7721 cells stably expressing POH1-Flag using anti-POH1(c) and anti-Flag antibodies (d), respectively. Nuclear DNA was visualized using DAPI staining. Scale bar, 20 μm. (e) The nuclear (Nu) and cytoplasmic (Cy) fractions of SMMC-7721 cells transfected with either control or the POH1 siRNA were extracted and analysed using the indicated antibodies. (f,g) SMMC-7721 cells stably expressing the siRNA-resistant forms of POH1-Flag or of POH1(NES)-Flag were analysed by immunoblotting using the indicated antibodies (f); cells were transfected with control or POH1 siRNA for 72 h, and the cell extracts were then analysed by immunoblotting (g). (h) SMMC-7721 cells expressing two different forms of POH1 were transfected with control or POH1 siRNA. 48 h after transfection, the cells were cultured for 10 days. The cells were stained with crystal violet. (i) The nuclear (Nu) and cytoplasmic (Cy) fractions of SMMC-7721 cells transfected with control or the nuclear-localized POH1. The cell extracts were then analysed by immunoblotting.