Effects of 'glucose deprivation' on tumor proliferation, response to rapamycin and signaling. A) Tumor proliferation rates based on BrdU incorporation. BrdU (50 μg/g) was injected intraperitoneally once daily for two days before sacrifice. Histologic sections were stained with anti-BrdU antibodies. For each sample, > 1,000 tumor cells were counted to determine the fraction of tumor cells with positive nuclear BrdU staining. *, p < 0.05. B) Response of the LEF2 tumors to rapamycin treatment (Rapa, 1 mg/kg - 5 days a week for 2 weeks) based on change in tumor volume. Three mice per group. C) Representative photomicrographs of LEF2 xenografts following 2-DG and rapamycin (Rapa) treatment. Magnification 400×. Note differences in cell size. D) Immunoblot of representative tumor lysates from the 4 treatment groups (2 each) showing the expression of the indicated proteins. W, Western diet; CF, carbohydrate-free diet. E) Western blot of tumor lysates from (B) showing effects of rapamycin on mTORC1 (p-S6), Akt (p-Akt), and autophagic (p62) pathways.