Figure 1

Effects on S100A4 expression and MTX sensitivity upon pCMV-S100A4 transfection of HT29 cells. A) mRNA levels of S100A4 determined by RT-Real-Time PCR 48 h after treatment of HT29 cells (30,000) with 250 ng of the expression vector for S100A4 (pCMV-S100A4). B) A representative image of the intracellular protein levels of S100A4 determined by Western Blotting 72 h after ectopic transfection with its expression vector is shown in the upper panel, and the quantification of the blots is shown in the lower panel. Purified S100A4 protein was used as a reference marker (Abnova; first lane). An additional panel showing endogenous S100A4 protein levels in HT29 sensitive (S) and resistant (R) cells is also provided. C) Effects of S100A4 overexpression on cell viability. HT29 cells (100,000) were treated with 1 μg of pCMV-S100A4 and 5 × 10^-8M MTX was added 48 h later. Cell viability was assessed by the MTT assay six days after MTX treatment. D) Extracellular S100A4 protein levels quantified by ELISA 72 h after S100A4 overexpression upon pCMV-S100A4 transfection. The expression and viability results are expressed as percentages referred to the untreated cells. Values are the mean of three independent experiments ± SE. *p < 0.05, **p < 0.01, *** p < 0.001.