Supplementary Figure 6: Induction of the AARE-Driven Expression system by halofuginone in liver and pancreas.

Halofuginone (Hf) exerts its effects by acting as a high affinity inhibitor of the enzyme Glutamyl-Prolyl tRNA synthetase. Inhibition of prolyl tRNA charging leads to the accumulation of uncharged prolyl tRNAs, which serves as a signal to initiate the amino acid starvation response. Hf has anti-metastatic and anti-proliferative effects. It has already been used to treat patients with solid tumor, unfortunatly with low antitumor effects and few side effects (nosea, vomiting, fatigue…) (De Jonge et al 2006; European J of Cancer 42; 1768-1774). (a) Bioluminescence imaging of mice following hydrodynamics-based DNA injection of the pGL3-AARE-Luc. Wild type mice received 25 micrograms of plasmid according to the hydrodynamic injection method. Twenty-four hours later, mice were injected with Hf (0.05μg/g of body weight) or PBS (Ctrl). Bioluminescence imaging was performed 6 hours after injection and light emission quantified using ROIs covering the abdominal area (red shape) (Student's t-test: ***p <0.001 versus Control (Ctrl), n=6 male mice; error bars: means ± s.e.m.). Signal intensity as a result of photon detection is graded from red (highest number of photons) to blue (lowest intensity). Next the livers have been collected and imaged for bioluminescence and then the corresponding protein homogenates were assayed for luciferase activity determination (Student's test: ***p <0.001 versus Control diet, n=6 male mice; error bars: means ± s.e.m.). (b) Intra-pancreatic delivery and induction of the AARE-Luc by halofuginone following lentiviral transduction. Administration of lentiviral particles containing the AARE-TK-Luc (LV-AARE-Luc) was performed into the pancreas of wild type mice. Ten days after injection, mice were injected with halofuginone (Hf) or PBS (Ctrl). Light emission was quantified 6 h after injection using ROIs covering the pancreatic area (red shape). Signal intensity as a result of photon detection is graded from red (highest number of photons) to blue (lowest intensity). (Student's t-test: **p <0.01 versus Control (Ctrl), n=6 male mice; error bars: means ± s.e.m.). Then the pancreas have been collected and imaged for bioluminescence and the corresponding protein homogenates were assayed for luciferase activity determination (Student's t-test: ***p <0.001 versus Control (Ctrl), n=6 male mice; error bars: means ± s.e.m.).