Figure 1

AMPK maintains cell survival under metabolic stress by promoting metabolism of non-glucose carbon sources. (a) Images representing the cellular morphology of H1299 cells stably transfected with either pCDNA3 vector (H1299-EV) or with dominant-negative form of AMPK-α1 subunits tagged with Myc (H1299-DN). These cells were grown under glucose-limiting conditions (1 mM glucose, 2 mM glutamine and 10% FBS) in the presence or absence of 0.5 mM AICAR (AIC) or 20 nM rapamycin (Rapa) for 36 h. (Scale bar, 50 μm). (b) Viability of H1299-EV and H1299-DN cells cultured under the conditions mentioned in a for 36 h, as assessed by PI staining. (c) H1299 cells were transfected with AMPK-α1/2-specific siRNA and grown under conditions used in b, viability of these cells was assessed by PI staining. (d) Viability of AMPK-α1/2^+/+ (WT) and AMPK-α1/2^−/− (DKO) MEFs cultured under the conditions mentioned in a for 36 h, as assessed by PI staining. (e) Representative images showing cellular morphology of WT and AMPK-DKO MEFs cultured in DMEM with or without glucose and FBS in the presence or absence of 0.5 mM AICAR or 20 nM rapamycin for 36 h (Scale bar, 50 μm). (f) H1299-EV and H1299-DN cells were grown in either 25 or 1 mM glucose in the presence or absence of 0.5 mM AICAR for 24 h. Representative immunoblots showing the levels of indicated molecules. Antibody for Myc tag (9E10) was used for detecting overexpressed dominant-negative form of AMPK α1 subunit. HSP60 was used as a loading control. (g) Representative immunoblots showing phosphorylated and basal levels of AMPK in H1299 cells transfected with either control or AMPK-α1/2-specific siRNAs. HSP60 was used as loading control. (h) Survival and (i) ATP level in H1299 cells (transfected with AMPK-α1/2 siRNA) cultured in glucose and glutamine-free DMEM supplemented with 5 mM glucose (Glu), 5 mM glutamine (Gln), 5 mM galactose (Gal), 10 mM lactate (Lact) and 100 μM palmitate (Palm) in the presence or absence of 0.5 mM AICAR (AIC), 10 μM compound C (CC) and 1 μM oligomycin (Oli) for 48 h. (j, k) Survival and relative ATP level in WT and DKO MEFs cultured under similar conditions used in h and i. Values are represented as mean±S.D. *P<0.05, **P<0.01, ***P<0.001 denote significant differences between the groups.