Fig. 5. Insulin treatment provokes an increase in Sima protein levels; involvement of TOR in the HRE response. (A) Sima protein levels upon insulin treatment. S2 cells were treated with insulin (10 µg/ml) (I) or exposed to hypoxia (2% O₂) for 16 hours (H) and Sima protein levels in nuclear extracts were analyzed by western blot (upper panel), hsp70 (lower panel) was used as a loading control. Sima protein levels were strongly upregulated upon insulin treatment, attaining levels similar to those caused by exposure to 2% O₂ during 16 hours (results of a representative experiment (n=3) are shown). (B) sima mRNA upon insulin treatment. Total RNA from cells treated or not with insulin (10 µg/ml) was analysed by northern blot using a specific sima probe (upper panel), S18 rRNA was used as a loading control (lower panel). sima mRNA levels did not change upon insulin treatment. (C) Effect of TOR on insulin-dependent induction of HRE-Luc. Cells were treated or not with insulin (10 µg/ml) for 20 hours in the presence of rapamycin (200 nM). Luciferase activity was determined and normalized to protein concentration; rapamycin provoked clear inhibition of the of HRE-Luc induction. (D) Silencing of dTOR, dRheb or dS6K. Cells were incubated in the presence of dsRNA directed against GFP, dTOR, dRheb or dS6K and 3 days later the cells were treated or not with 10 µg/ml insulin for 20 hours, then lysed and luciferase activity was determined; dTOR, dRheb and dS6K dsRNAs provoked strong inhibition of HRE induction.