Figure 3

Effect of SMC3 overexpression on 293 cells colony growth. a): 293 cells at 70% confluence were transfected with 1 μg/ml of pcDNA3.1 (control) or SMC3-pcDNA3.1 expression vectors and cell RNA collected after 48 hrs in 1 ml TriReagent. SMC3, and G3PDH transcript levels were assayed by semiquantitative RT-PCR and the products analyzed by agarose electrophoresis. The result of a control and two independent samples of cells transfected with SMC3-pcDNA3.1 are shown. b) Growth rate of cells overexpressing SMC3. Cells were seeded in 96-well plates (2,500 cells/well in a final volume of 150 μl) in DMEM medium supplemented with 1.5% FCS. Cell proliferation was assessed by using a CellTiter 96 colorimetric assay. Each point represent the mean ± SD of four independent determinations. c,d) Transfected cells were seeded in 35 mm plates and grown for 10 days in DMEM supplemented with 1.5% FCS. To detect foci of transformation, cells were fixed in 70% ethanol followed by staining with 0.1% methylene-blue. e,f) Colony formation in semisoft agarose. Twenty-four h after transfection, cells were trypsinized and resuspended in 0.2% agarose in DMEM containing 10% fetal bovine serum and plated on top of solidified agarose (0.4%) dissolved in the same medium in 35 mm dishes. After 3 weeks of culture cell colonies were examined under a light microscope.