Fig. S1 1 Oxygen consumption rate (pmol/sec/10⁶cells) shLKB1#1 control * untreated 25 μM erlotinib H358 Vector LKB1 Oxygen consumption rate (pmol/sec/10⁶cells) untreated 25 μM erlotinib H460 * Figure S1. Erlotinib inhibits oxygen consumption rate (OCR) in LKB1-deficient cells. A, OCR of LKB1 stable knockdown and non-targeted shRNA control H358 cells. B, OCR of H460 cells expressing empty pBABE vector or wild-type LKB1. Cells were treated with erlotinib for 24 h. Data are mean ± SEM (n=3). *; p<0.05, untreated/erlotinib. A B

A Specific growth rate (h⁻¹) H460 VectorLKB1 B Fig. S2 Figure S2. Effect of erlotinib on growth and glycolytic metabolism of H460 cells. A, Effect of treatment with erlotinib on cell growth rate in H460 cells expressing empty pBABE vector or wild- type LKB1. Cells were seeded in 96-well plates. After 24 h, cells were treated with the indicated concentrations of erlotinib and growth was monitored for 72 h. B, Glucose uptake rate and lactate production rate in H460 cells expressing empty pBABE vector or wild-type LKB1. Cells were grown for 72 h in the presence of treatments. 2 VectorLKB1 Glucose uptake rate (µmol/10⁶ cells/h) Lactate production rate (µmol/10⁶ cells/h) VectorLKB1 untreated 15 μM erlotinib untreated 15 μM erlotinib H460

Fig. S3 p-AMPKα AMPKα p-ACC ACC control erlotinib hours shLKB1 # H358 actin Figure S3. Erlotinib-induced activation of AMPKα in LKB1-deficient H358 cells. Erlotinib activated AMPKα and induced phosphorylation of ACC in shLKB1 H358 cells. Following treatment with 0.5 μM erlotinib for the indicated time, cell lysates were analyzed by Western blotting. The expression of phosphorylated AMPKα was calculated as the ratio of phosphorylated AMPKα to total AMPKα protein expression using densitometric analysis. The ratio of phosphorylated to total ACC was calculated similarly. All expression ratios were normalized to the untreated group.

Fig. S4 control shLKB1#1 0.5 µM erlotinib -+ A -+ p-mTOR (Ser2448) p-AMPKα mTOR AMPKα p-p70S6K (Thr389) p-Akt (Ser473) Akt p-Erk B H358 A vector LKB1 p70S6K (Thr389) P-4EBP1 (Thr37/46) P-4EBP1 actin Erk vector LKB1 + + H µM erlotinib p-mTOR (Ser2448) p-AMPKα mTOR AMPKα p-p70S6K (Thr389) p-Akt (Ser473) Akt p-Erk p70S6K (Thr389) P-4EBP1 (Thr37/46) P-4EBP1 actin Erk Figure S4. Erlotinib-induced activation of AMPKα selectively inhibits mTOR signaling pathway in LKB1-deficient cells. Erlotinib activated AMPKα and blocked mTOR pathway signaling in A, shLKB1 H358 cells and B, LKB1-non expressing A549 and H460 cells. After 24 h of 0.5 μM erlotinib treatment, cell lysates were analyzed by Western blotting using specific antibodies for the indicated proteins. Blockade of mTOR activity following erlotinib treatment resulted in decreased phosphorylation of p70S6K and p-4EBP1 in LKB1 deficient cells.

Fig. S5 control shLKB1#1 0.5 µM erlotinib-+ shLKB1#2 A -+-+ PDK4 Actin Calu-6 A µM erlotinib -- vector LKB1 PDK4 Actin ++ control shLKB1#1 0.5 µM erlotinib -+-+ PDK4 Actin H358H µM erlotinib -- vector LKB1 PDK4 Actin ++ Figure S5. Levels of PDK4 expression is not changed by erlotinib treatment in both LKB1-deficient and overexpressing NSCLC cells. A, Levels of PDK4 expression in LKB1 stable knockdown and non- targeted shRNA control Calu-6 and H358 cells. B, Levels of PDK4 expression in A549 and H460 cells expressing empty pBABE vector or wild-type LKB1. Cells were treated with 0.5 μM erlotinib for 24 h and cell lysates were analyzed by Western blotting. B