The role of the conserved regions -74/-65 and -62/-48 in the 34-nt region in transcriptional activity in HepG2 cells. (A) Expression of edn and ecp mRNA in HepG2 and HL-60 clone 15 cells. Total RNA of HepG2 and HL-60 clone 15 were isolated and reverse-transcripted into cDNA. Specific primers for ecp or edn were used to amplify the cDNA. The PCR products were electrophoresed on a 2.0% agarose gel and stained with ethidium bromide. (B) HepG2 cells were transfected with the luciferase reporter plasmid pGL3 basic or the same vector containing edn or ecp upstream sequences with or without the 34-nt segment. (C) HepG2 cells were transfected with the luciferase reporter plasmid pGL3 basic or pGL3-edn, pGL3-edn Δ (-81/-48), pGL3-edn mut (-74/-65), pGL3-edn mut (-62/-48), respectively. The promoter activities were measured using the luciferase assay system. The average values of promoter activities were calculated as described in Methods and obtained from five independent experiments. The difference between the two groups is statistically significant (P < 0.05), as determined by the Wilcoxon Rank Sum test.