Fig. 2. Insulin induces an HRE-response in S2 cells. (A) Effect of insulin on HRE-Luc reporter induction. S2 cells stably transfected with HRE-Luc were cultured for 3 days and then exposed or not to hypoxia (1% O₂), DFO (100 µM) or insulin (50 µg/ml) for 20 hours. Luciferase activity was determined and normalized to protein concentration. HRE-Luc was induced by insulin, and attained levels similar to those upon exposure to hypoxia and higher than those observed upon DFO treatment. (B) Treatment with different doses of insulin. Cells were stimulated with 1-50 µg/ml insulin for 20 hours, lysed and luciferase activity was determined and normalized to protein concentration. HRE-Luc was induced as a function of insulin concentration. (C) Endogenous dLDH mRNA levels upon insulin treatment. To assess the effect of insulin on the expression of the endogenous dLDH gene, cells were treated for 4, 8, or 16 hours with insulin (10 µg/ml) and then total RNA was prepared and analyzed by northern blot using a specific dLDH probe (upper panel) or 18S rRNA as a control (lower panel); expression of dLDH mRNA increased as a function of the time of exposure to insulin. (D) Role of Sima and Tango in insulin-dependent induction of HRE-Luc. Cells were incubated in the absence (control) or presence of dsRNA directed against GFP, sima or tango and 3 days later, they were treated or not with 10 µg/ml insulin for 20 hours. Cells were then lysed and luciferase activity determined. Induction of the HRE reporter by insulin was totally abrogated by sima or tango dsRNA.