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. 2012 Jul 18;7(7):e40955. doi: 10.1371/journal.pone.0040955

Figure 5. Overexpression and silencing of DKK1 in SW480 cells.

Figure 5

pCMV vector containing human DKK1 gene was used to transfect cells before sample collection for analysis. Empty pCMV vector was used as the control for the overexpression experiments. Three independent cell samples were analyzed and presented as the mean ± SEM. A) mRNA expression of DKK1 and Cyclin D1 in regular and DKK1-transfected SW480 cells. The mRNA expression was analyzed by RT-PCR. Data were normalized to internal control L7a. B) DKK1 protein expression in regular cells and transfected SW480 cells. Whole cell protein extracts were collected from transfected SW480 cells and western blot analysis of DKK1 was performed as described in materials and methods. A representative blot is shown and the quantification represents the mean± SEM from 3 independent dishes. Actin was used as the loading control. C) Cell cycle analysis of regular and DKK1-transfected SW480 cells. Result was obtained by flow cytometry. Y-axis represents % of gated cells and x-axis shows different cell cycle stages, including G1, S and G2/M. D) WST-1 proliferation assay in vector- and DKK1-transfected SW480 cells. WST-1 signals were converted to actual cell numbers using a standard generated by serial dilutions of a known number of cells. Data were normalized to control for genistein treatment and vector for DKK1 transfection, respectively. Asterisks (*) indicate statistical significance compared with the respective control group (genistein to control; DKK1 to vector; p<0.05). For the knockdown of DKK1, siRNA duplexes against human DKK1 gene were transfected into SW480 cells. Scrambled sequences were used as the control siRNA. E) mRNA expression of DKK1 and Cyclin D1 in control siRNA (N/S si) and DKK1 siRNA (DKK1 si)-treated SW480 cells by control and genistein treatments. The mRNA expression was analyzed by RT-PCR. Data were normalized to internal control L7a. F) WST-1 proliferation assay in control siRNA and DKK1 siRNA SW480 cells. WST-1 signals were converted to actual cell numbers using a standard generated by serial dilutions of a known number of cells. Asterisks (*) indicate statistical significance compared to Control 0 µmol/L genistein (p<0.05). The bracket indicates statistical difference between the N/S si and DKK1 si groups treated with genistein (p = 0.002).