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. 2014 Jan 13;111(4):E435–E444. doi: 10.1073/pnas.1311121111

Fig. 6.

Fig. 6.

Metformin suppresses proliferation through PRAS40-mediated mTOR inhibition. (A and B) Immunoblot using mTOR and 4EBP1 antibodies showing shRNA-mediated knockdown of mTOR and 4EBP1 in T98G glioma cells. Actin was used as a loading control. (C) Proliferation of control (NT) and mTOR shRNA-expressing and 4EBP1 shRNA-expressing T98G cells treated with AICAR and metformin. (D) Immunoprecipitation of RAPTOR followed by immunoblot analysis showing the effect of metformin and other cell stressors on RAPTOR–PRAS40 association. Loading control lysates are shown in the bottom three panels. (E) Proliferation of metformin-treated glioma cells expressing NT or PRAS40 shRNA. (E″) Immunoblot showing PRAS40 knockdown. (F) The model shows active AMPK is highly expressed in glioblastoma. The direct AMPK activator A769662 or the indirect activator AICAR does not effectively suppress mTOR. Although A769662 has no effect on glioma proliferation, AICAR suppresses glioma proliferation by degrading a crucial G2M phosphatase cdc25c through the proteasome, independent of AMPK. The other AMPK agonist metformin represses glioma proliferation through mTOR inhibition by increasing PRAS40-RAPTOR interaction, independent of AMPK. Data are representative of two to four independent experiments. *P ≤ 0.001. Error bars in C and E represent mean ± SD.