The LKB1/AMPK signaling pathway in AML is functional and controls mTOR activity. (A) Western blot analysis of the phosphorylation levels for AMPK_α T¹⁷², ACC S⁷⁹, mTOR S²⁴¹⁸, P70S6K T³⁸⁹, 4E-BP1 T^37/46, 4E-BP1 S⁶⁵, 4E-BP1 T⁷⁰, and p42/44 ERK T²⁰²/Y²⁰⁴ in primary AML samples and in the MV4-11 and K562 leukemic cell lines cultured for 24 hours without or with 5, 10, or 15mM metformin. (B) Western blot analysis of the phosphorylation levels of AMPK_α T¹⁷², ACC S⁷⁹, mTOR S²⁴⁸¹, P70S6K T³⁸⁹, 4E-BP1 T^37/46, and 4E-BP1 S⁶⁵ in the K562 leukemic cell line, expressing the AMPK^R69Q activated mutant after adenoviral infection. AMPK^R69Q is detected with an anti-AMPK_γ antibody. (C) Quantification of Western blot signals. The ratios of phospho-P70S6K T³⁸⁹, phospho-4E-BP1 S⁶⁵, phospho-4E-BP1 T^37/46, or phospho-AMPK T¹⁷² to the actin signal intensity were calculated, and results were expressed relative to the control conditions (without metformin) in each experiment. Each histogram represents the mean of 25 independent experiments using primary AML samples, and vertical bars indicate the SEM.