Fig. 1. Hypoxic induction of an HRE-response in a stably transfected Drosophila S2 cell line. (A) Schematic representation of the HIF-responsive firefly luciferase reporter gene used in this study (HRE-Luc), in which transcription depends on a dimerized 51 bp sequence from the murine lactate dehydrogenase enhancer that contains two HIF responsive elements (HREs) and one cyclic AMP responsive element (CRE). (B) Effect of hypoxia on HRE-Luc activity in S2 cells. Cells were exposed to different oxygen levels for 20 hours or stimulated with 100 µM DFO. Luciferase activity was determined and normalized to protein concentration in the extracts. Induction of the reporter augmented when oxygen levels decrease. (C) Variations in dLDH mRNA levels. To measure the effect of hypoxia on endogenous dLDH gene expression, cells were exposed to 2% O₂ for 16 hours. Total RNA was then prepared and analyzed by northern blot. LDH mRNA is strongly induced by hypoxia (upper panel). 18S rRNA that was used as loading control remains constant (lower panel). (D) Role of Sima and Tango in HRE-Luc induction. Cells were incubated with dsRNA directed against GFP (control), sima or tango and 3 days later, the culture was treated or not with DFO (100 µM) or was exposed to 1% O₂ for 20 hours. Cells were then lysed and luciferase activity was determined. Hypoxia or DFO-dependent induction of the reporter was specifically abrogated by sima or tango RNAi treatment. (E) Effect of exogenous Sima. Cells were transiently transfected with an empty vector (pAC) or with a plasmid expressing Sima protein (pAC-Sima), together with 100 ng of a vector encoding Renilla luciferase (pRL). Twenty-four hours after transfection, cells were lysed and firefly or Renilla luciferase activity was determined. Induction of the HRE-Luc reporter was proportional to the transfected amount of sima-encoding plasmid. Normalized luciferase activity (firefly/Renilla ratio) is expressed as fold-induction with respect to the activity upon transfection of an empty vector. In all experiments involving luciferase determinations, data represent the mean±s.e.m. of three independent experiments performed in triplicate and expressed as fold-induction with respect to untreated cells.