Figure 7. Increased cell growth by CDO1 knockdown.

A, siRNAs targeting CDO1 mRNA (siR-1 ∼ -4) and a non-targeting control siRNA (siR-Cont) were transfected into CDO1-expressing HEK293 cells, and CDO1 gene knockdown was examined by RT-PCR analysis (left) and cell growth was determined by the MTT assay (right). β-actin was used as a loading control. Two independent experiments were done in triplicate, and values are expressed as means ± SD. *, P<0.05 in T-test. Both siR-1 and 2 reduced the CDO1 mRNA, but only siR-2 displayed increased HEK293 cell growth (left). B, The morphology of HEK293 cells were examined under a phase-contrast microscope after siRNA-transfected cells were grown on Matrigel beds for 3 days. MG alone, a picture of Matrigel without cells. Scale bar, 500 µm. C, HepG2 cells express CDO1, but do not harbor the gene methylation (data not shown). CDO1 siRNA (−1 and −2) or control siRNA were transfected into HepG2 cells, and RT-PCR and MTT assays were performed. N, cells without transfection. Increased cell growth was observed in cells transfected only with siR-2. D, Colony focus assays were performed in HepG2 cells after transfection with siR-2 and controls. Colonies were grown for 13 days and stained with crystal violet solution. After air-drying, colonies were counted (left) and photographed (right). Two independent experiments were done in triplicate, and values are expressed as means ± SD. *, P<0.05 in T-test. E, The in vitro cell invasion assay was performed in the HepG2 cells after transfection with siR-2 and controls. Cells were incubated for 16 hrs, and after fixation and staining, invading cells were counted at 100 X magnification (left). Cell growth for 16 hrs determined by MTT assay was not significant (right).