Effects of energy stress on AMPK phosphorylation and apoptosis in CT-2A tumor and normal brain. a) Influence of 40% dietary caloric restriction on AMPK phosphorylation and caspase-3 cleavage in CT-2A tumor grown orthotopically in B6 mice and in contra-lateral normal brain tissue. Tumor implantation, diet administration and collection of tumor and brain tissues were described in methods. Tissue lysates were analyzed with indicated antibodies by western blot. Results indicate increased AMPK phosphorylation and caspase-3 cleavage in the tumors of CR mice than in AL mice. Normal brain had much less AMPK phosphorylation compared to CT-2A tumor. Tissues from 3 independent mice for each group were analyzed. b) Ratio of AMPK phosphorylation to total phosphorylation. Asterisk indicates a significant increase in AMPK phosphorylation at p < 0.01. c) Influence of 40% dietary caloric restriction on apoptotic cells in CT-2A tumor grown orthotopically. Tumor implantation and diet treatment was the same as mentioned above. After 11 day, brain was dissected out and fixed in formalin. Paraffin embedded tissue was processed for TUNEL staining. Results indicate that the TUNEL positive apoptotic cells (black arrows) are higher in CR tumor than AL tumor. Surrounding normal brain has no significant cell-death. d) Tissues from 3 independent mice for each group were analyzed. Apoptotic cell index was significantly higher in CR tumor at p < 0.0001.