Loss of LKB1 sensitizes cells to metabolic stress. (A) Immunoblotting was performed on protein extracts from NIC-FF and NIC-LKB1 KD cells treated with rapamycin (100 nM) for 24 hours to confirm mTOR activity. Arrows point to the hypophosphorylated forms of 4E-BP1. (B-C) ECAR analysis of NIC-FF and NIC-LKB1 KD cells treated or not with rapamycin for 24 hours in full glucose conditions (B) or in 1 mM glucose conditions (C). (D) ECAR analysis of NIC-FF and NIC-LKB1 cells treated with vehicle or with metformin (5 mM) for 6 hours. (E-F). Viability assays were performed on NIC-FF and NIC-LKB1 KD cells cultured in 25 mM glucose or 1 mM glucose and treated or not treated with rapamycin for 72 hours. The level of apoptosis was assessed using 7-AAD (E) or cleaved caspase 3 levels (F). All graphs correspond to a representative experiment of three performed, and values represent the average of six wells for (B), (C), (D) and (F) and three wells for (E). (G) NIC-FF and NIC-LKB1 KD cells were cultured for 24 hours in 1 mM glucose and intracellular ATP levels were quantified as the percentage drop from baseline conditions (25 mM of glucose). *, P< 0.05; **, P< 0.01; ***, P< 0.001.