Sickle mononuclear leukocytes up-regulate E-selectin mRNA in HUVEC.

HUVEC were incubated with sickle or normal mononuclear leukocytes (2 leukocytes/endothelial cell) for 5 hours or TNF-α (10 ng/mL) for 2 hours. E-selectin mRNA was isolated from the endothelial cells, reverse transcribed into cDNA, and amplified by PCR as described in “Materials and methods.” RT-PCR products were separated by electrophoresis on a 2% agarose gel and stained with ethidium bromide. The figure is a reverse image of the stain. Lane 1, 250-bp DNA ladder; lane 2, control (media only) HUVEC; lane 3, TNF-α–treated HUVEC; lane 4, normal mononuclear leukocyte-treated HUVEC; lane 5, sickle mononuclear leukocyte-treated HUVEC. GAPDH mRNA, a control, was also amplified by RT-PCR from the same RNA extracts and run on a separate agarose gel shown at the bottom. The E-selectin and the GAPDH products measured 1000 bp and 600 bp, respectively, as predicted from the gene sequences. Results are from a single representative experiment of 2.