Determination of mRNA levels and cytotoxicity upon siRNA treatment of HT29 sensitive cells. (A) The mRNA levels of CAV1, ENO2, PKCα and DHFR were determined by RT-Real-time PCR 48 h after treatment of HT29 cells with the indicated concentrations of siCAV1, siENO2, siPKCα, siDHFR and a non-related siRNA. Symbols used for each mRNA are presented as an insert within the figure. (B) Cells were treated with 100 nM of each siRNA as previously described and 5 × 10-8 M MTX was added after 48 h. Cell viability was determined after 5 days from the beginning of the treatment. All results are expressed as percentages referred to untreated cells. Values are the mean of three independent experiments ± SE. A non-related (NR) siRNA was used as negative control.