Fig. 2. A Tax-responsive element located at −962 to −873 of the promoter region of eNOS is involved in increasing eNOS mRNA transcription in aphidicolin-treated BAECs. (A) After BAECs were treated with 20 µM aphidicolin for the indicated times (0, 8, 24, or 48 h), total RNA were extracted and cDNA were synthesized via a reverse transcription reaction. Level of eNOS mRNA expression was determined using quantitative real-time PCR (qPCR) as described in the MATERIALS AND METHODS. (B) BAECs were treated with various doses of aphidicolin (0, 10, 20, or 40 μM) for 24 h and the level of eNOS mRNA expression was determined using qPCR as described in
Fig. 2A. (C) A schematic illustration of the eNOS gene promoter from −1600 to −428. The eNOS gene promoter contains an Sp1/AP2 site between −1600 and −962, a TRE site between −962 and −873, and a CRE site between −873 and −428. (D) After 5’-serially deleted eNOS promoter cDNAs fused into a luciferase gene reporter plasmid were transfected into BAECs, the cells were incubated for 24 h in the absence or presence of 20 µM aphidicolin. Cells were harvested and luciferase activity was determined as described in the MATERIALS AND METHODS. All experiments were performed at least four times independently (n=4). Bar graphs depict mean fold alterations above the controls (± SD). Statistical significance was evaluated using either Student’s
t test or ANOVA. All differences were considered to be statistically significant at a
p value of <0.05. **
p<0.01. n.s., not significant.
