Figure 5.

mTORC1 inhibitor rapamycin inhibits cell proliferation and metastasis, suppresses the mTORC1 pathway and activates the AMPK pathway. (A) BL6-10 tumor cells cultured in flasks at 1 g or 1 g + rapamycin were counted daily for three days to measure cell proliferation. (B) BL6-10 tumor cells cultured at 1 g and 1 g + Rapa for three days were i.v. injected into C57BL/6 mice. Mouse lungs were collected 21 days after tumor cell injection, and black tumor lung colonies were counted. (C) Lysates prepared from BL6-10 cells cultured for 3 days at 1 g or 1 g + rapamycin were subjected to SDS-PAGE analysis. Proteins were transferred onto PVDF membranes and blotted with indicated antibodies. Western blot band signals were quantified by chemiluminescence. Densitometric values were normalized to matching GAPDH control. Data represent the mean ± SD of three independent experiments. *p < 0.05 versus indicated groups. (D,E) BL6-10 cells cultured for 3 days under 1 g or 1 g + rapamycin were subjected to mitochondria biogenesis assay using MiltoTracker Green kit. Cellular mitochondria biogenesis was quantified by flow cytometry (D). MFI: mean fluorescence intensity. Cellular mitochondria biogenesis was measured by confocal microscopy (E). Scale bar: 20 µm. (F) BL6-10 cells cultured for 3 days under 1 g or 1 g + Rapa were subjected to cell glycolysis assay using pH-Xtra^TM Glycolysis Assay kit. One representative experiment of two is shown.