The SHG44-DKK-1 cells appeared similar to the non-transfected cells and sometimes formed

clusters (Fig. 1c, d). Figure 1 Microscopic images of different groups cells in selection. Normal SHG44 (1a), normal SHG44 cells cultured in the presence of G418 for two weeks (1b); and SHG44-DKK-1 cells cultured in the presence of G418 for three weeks (1c, 1d). PCR analysis of DKK-1 in SHG44 cells DNA was extracted from cells of normal SHG44, see more SHG44-EV and SHG44 -DKK-1. The extracted DNA was amplified by PCR using the primer pair described above. As expected, a 223bp fragment was observed in SHG44 -DKK-1cells, but not in normal SHG44, or SHG44 -EV cells (Fig. 2). This result further confirmed the specific AC220 transfection of DKK-1 gene into the SHG44 cells. Figure 2 PCR amplification of DKK-1 SHG 44 -DKK-1 cells was lane 1, SHG 44 -EV was lane 2, normal SHG 44 cells was lane 3 and control (culture medium only) was lane 4. M was the marker for standard DNA molecular mass. DKK-1 mRNA expression in SHG44 cells RNA extracted from normal SHG44, SHG44-EV and SHG44 -DDK-1 cells was amplified by RT-PCR and subsequently analyzed by DNA gel. A prominent 223 bp band was detected from SHG44 -DKK-1 cells, but non-detectable

from SHG44 -EV cells or normal SHG44 cells (Fig. 3). Figure 3 RT-PCR analysis of DKK-1 mRNA expression. HDAC inhibitor Lane 1, 3 and 5 β-actin from cells of SHG44-DKK-1, SHG44-EV and normal SHG44 respectively. Lane 2, 4, 6 were DKK-1 mRNA from cells of SHG44-DKK-1, SHG44-EV and normal SHG44 respectively. M was the marker of standard DNA molecular mass. DKK-1 protein expression in SHG44 cells The total protein 3-mercaptopyruvate sulfurtransferase exacted from normal SHG44, SHG44-EV and SHG44 -DDK-1 cells was separated using 12% SDS-PAGE and was subsequently analyzed by Western

blot. A 35KD band, which corresponds to the size of DKK-1 protein was observed in SHG44 -DKK-1 cells, but not in SHG44 -EV or normal SHG44 cells (Fig. 4). Figure 4 Western blot analysis of DKK-1 protein. It showing DKK-1 protein from cells of normal SHG44 (lane 1), SHG44-EV (lane 2) and SHG44-DKK-1 (Lane 3). β-actin was used as loading control. BCNU induced apoptosis BCNU is an anti-cancer drug and an inducer of apoptotic cell death. We used BCNU to further assess the role of DKK-1 in SHG44 cells. Apoptosis was observed in all three groups of cells: normal SHG44, SHG44-EV and SHG44 -DDK-1. The average apoptosis ratio of normal SHG44, SHG44-EV cells and SHG44 -DKK-1, was2.5 ± 0.2%, 1.8 ± 0.2%, 8.4 ± 0.3%, respectively(Fig. 5). The apoptosis ratio of SHG44 -DKK-1 cells was significantly (P < 0.05) higher than that of normal SHG44 or SHG44-EVcells. Minimal apoptosis was observed in all three groups of cells in the absence of BCNU. Figure 5 Apoptosis ratio was detected by flow cytometry analysis. Representative image of flow cytometry analysis of BCNU treated cells, showing the apoptosis ratio (right lower-quadrant) of normal SHG44 (a), SHG44-EV (b) and SHG44-DKK-1 (c) cells.