Figure 3.

Acidosis increases glutaminolysis and renders glutaminolysis essential. (A) Heatmap shows the changes in the intracellular amino acids under acidosis (HCl), lactosis (10 and 25 mM Lac), or lactic acidosis (10 and 25 mM LA) (n = 3) conditions. (B) Normalized levels of intracellular glutamine (Gln) and glutamate (Glu) in MCF-7 cells under control or lactic acidosis conditions (n = 4). (C) Relative cell numbers as determined by trypan blue exclusion, of MCF-7 and ZR-75-1 cells after 72 h under the indicated media conditions. (n = 4) (D) Relative ATP levels under acidosis (Acid) with (+) or without (-) of glutamine (Gln) in media. (E,F) Relative changes of indicated mRNAs (E) and proteins (F) under indicated conditions. (G) Relative cell numbers of MCF-7 cells when transfected with control or two GLS2 small interfering (si)RNAs under control and acidosis conditions. (H) Total glutathione (GSH) level of MCF-7 cells exposed to acidosis or lactic acidosis (n = 3). (I) Normalized glutathione disulfide (GSSG)/GSH ratios for MCF-7 and ZR-75-1 cells under control, acidosis or lactic acidosis conditions (n = 6). (J) Normalized cell numbers of MCF-7 cells under control, acidosis and lactic acidosis conditions when exposed to indicated level (uM) of H₂O₂ (n = 3). (K,L) Relative cell numbers (via trypan blue exclusion) (K) and cellular ATP level for MCF-7 (L) treated with vector or 0.2 mM amino-oxyacetate (AOA) for 72 h. The indicated samples were supplemented with 700 μm α-ketoglutarate (α-KG). Error bars are mean ± SEM, P values as indicated (*P ≤0.05, **P ≤0.001, ***P ≤0.0001).