Figure 2.

Alcohol activates GILZ promoter via GRE cis-elements. (A) The gilz promoter was cloned into the promoter-less pGL.4.16[luc2CP/Hygro] construct, generating the pGL-Long-GILZp-Fluc plasmid. GRE3-5 sites were removed by deletion of the region (−1,560 to −1,884), resulting in the pGL-Short-GILZp-Fluc plasmid. The GRE1 and GRE2 sites were mutated, producing the pGL-Short-mutGILZp-Luc plasmid. (B) A549 cells were, respectively, transfected with one of the three resulting plasmids in combination with the reference plasmid pGL4.75-hRluc that constitutively expressed the humanized Renilla luciferase. Forty-eight hours after transfection, the cells were exposed to 0 or 50 mM alcohol or 1 µM of Dex for 24 h. Potency of the wild-type or each mutated gilz promoter to drive the reporter gene expression was determined by the activity of the Firefly luciferase that had been normalized to that of the Renilla luciferase in each sample. Asterisks denote significant differences between the comparing groups by Student’s t-test (*p < 0.05, **p < 0.01; n = 3).