Figure 3

Mutation analysis of GADD153 promoter induction by hypoxia. (A) Induction of luciferase activity in the H9c2cells in response to hypoxia. The cells were transiently transfected with GADD153 promoter-deletion constructs and then the cells were harvested for determination of the luciferase activity 24 h after hypoxia/reoxygenation treatment. The induction ratios are expressed relative to the untreated controls. The values are means ± SEs of three independent experiments. (B) Hypoxia-induced AP-1 activity. The cells were transfected with either the AP-1-responsive reporter construct (TRE)₄-TK-Luc or the empty vector and then they were cultured under normoxic or hypoxic conditions. Each bar represents the mean ± SE of two experiments performed in duplicate. (C) Hypoxia-induced CAT activity from GADD153 constructs containing wild type AP-1, deleted AP-1 mutant, or 3 × AP-1 (left). The H9c2cells were transiently transfected with the indicated CAT reporter gene constructs and cultured under hypoxia and reoxygenation as described in the Methods. Activation is expressed relative to mock control. Each bar represents the mean ± SE of two experiments performed in duplicate (right). P < 0.01.