Figure 9. Induction by MN10021 and analogs of mRNA for iNOS in HUVEC.

A. HUVEC were grown to 80–90% confluence in 12-well tissue culture plates using defined media provided by Cambrex and supplemented with 10% FBS. The cells were allowed to become quiescent by incubating them overnight in the basal media (without growth factors) supplied by Cambrex and supplemented with 1% FBS. Triplicate wells were incubated with either media alone or media containing 50 µM MN10021 for 3 h at 37°C, the wells washed with warm PBS, and the cells lysed. Total RNA was isolated, cDNA prepared, and RT-PCR performed as described in Materials & Methods. Primers for human iNOS and β-actin were obtained from R & D systems. B. HUVEC were prepared as described under Methods, grown to 80–90% confluence in 12-well tissue culture plates, and incubated overnight in basal medium (no growth factors) containing 1% FBS in order to make them quiescent. Triplicate wells were incubated for 3 h at 37°C with 50 µM of one of four different 8-amino acid long analogs of MN10021. DUK0001: NH₂-GLDLLFLK-COOH; DUK0004: acetyl-GLDLLFLK-acetyl; DUK0005: acetyl-GLDLLFLK-NH₂; DUK0006: NH₂-GLDLLFLK-NH₂. The wells were washed with warm PBS, and the cells lysed. Total RNA was isolated, cDNA prepared, and RT-PCR performed as described in Materials & Methods. Primers for human iNOS and β-actin were obtained from R & D systems. C. HUVEC were prepared and tested as described in panel A. The sequence of DUK0007 is acetyl-GLDLLYLK-NH₂ which differs from that of DUK0005 in that it has a Tyr at position 6 in place of a Phe.