Stimulation of endothelial cell migration and tubelike differentiation by the overexpression of 12-LOX in endothelial cells.

CD4 cells were transfected with a 12-LOX expression construct or an empty vector as a control. (A) Detection of 12-LOX mRNA expression by RT-PCR. Primers were the pair of the primers for the first round of PCR. The reaction was carried out for 30 cycles. Control, no RNA present in PCR or RT reaction mixtures as controls for the quality of PCR; RT(−), no reverse transcription present in RT reaction mixtures as controls for the possible contamination of DNA in RNA samples; RT(+), reverse transcription present. Vector, CD4 cells transfected with pcDNA 3.1; LOX, CD4 cells transfected with pcDNA construct with 12-LOX cDNA insert. (B) Northern blot analysis of 12-LOX mRNA levels. Control, loading buffer as the blank control. (C) Analysis of 12-LOX expression at the protein level by immunoblot. (D) Increased cell migration in CD4 12-LOX transfectants. The migration assay was performed as detailed in “Materials and methods” using CD4 cells transfected with pcDNA (open circle) or pcDNA 12-LOX construct (filled circle). Data point, mean from 30 fields counted; bars, SE. The migration assay was repeated for 3 times with similar results. (E) Increased formation of tubelike structures in CD4 12-LOX transfectants. Left panel, vector control; right panel, 12-LOX transfected CD4 cells.