Effects of Compound 1 on lactate production in hypoxia/anoxia and the role of lactate dehydrogenase B (LDHB) expression in breast cancer cells. (A) Effect of hypoxia on lactate production EC50 values of Compound 1. MDA-MB-453 cells were cultured in normoxic (21% oxygen) or hypoxic (1%) conditions overnight. Medium was exchanged with physiological DMEM containing dimethyl sulfoxide (DMSO) or Compound 1 at multiple concentrations and collected after 2 h for hypoxic cells and 6 h for normoxic cells. EC50 values were estimated based on a 50% reduction in lactate production (dotted lines). (B) Extracellular acidification rate (ECAR) as a measure of lactate production after mitochondrial inhibition was quantified for MDA-MB-453. The baseline ECAR reading was obtained, and multiple concentrations of Compound 1 (2.5 to 40 μM) or DMSO were added followed by rotenone (Rot) (1 μM) and antimycin (1 μM). ECAR reading immediately prior to Compound 1 injection was set at 100%. (C) ECAR response of MDA-MB-453 cells to rotenone/antimycin after Compound 1 addition from (B) as a function of Compound 1 concentration. The averages of the final three readings were normalized to the untreated DMSO control. EC50 value was estimated based on a 50% reduction in ECAR. (D) Ssensitivity of a panel of breast cancer cell lines to Compound 1 after mitochondrial inhibition. Log2-linear slopes of ECAR response (obtained as in (C) for MDA-MB-453) were estimated for multiple cell lines between 2.5 and 40 μM. Means ± standard error (SE) of fitted values are shown. Cells were classified as low LDHB if the primary LDH tetramer was LDH5. Relative LDHB expression was determined previously by non-denatured electrophoresis . (E) Effect of LDHA or LDHB expression on Compound 1 sensitivity in HCC1937 cells. Stable isogenic lines were created using a lentiviral shRNA (non-silencing. LDHA, or LDHB) with puromycin selection.