LKB1-deficient breast cancer cells display increased aerobic glycolysis. (A-B) Extracellular flux analysis of NIC cell lines. NIC-FF and NIC-LKB1 KD cells were plated for ECAR (A) and OCR (B) analyses. The data represent one representative experiment of three independent replicates and the values correspond to an average of five wells per experiment. (C-D) NIC-FF and NIC-LKB1 KD cells were cultured for 48 hours and the relative extracellular (C) and intracellular (D) levels of lactate were determined using an enzymatic assay and GC-MS, respectively. The data represent one representative experiment from three independent replicates, each performed in triplicate. (E) Total RNA was isolated from NIC-FF and NIC-LKB1 KD cells, and the relative mRNA expression of several glycolytic enzymes was determined by qPCR. Transcript levels were determined relative to Rpl13 (60S ribosomal protein L13) mRNA levels, and normalized relative to its expression in control NIC-FF cells. The data represent the average of three independent experiments, each performed in triplicate. (F) Glucose uptake in NIC-FF and NIC-LKB1 KD cells was measured by flow cytometry using the 2NBDG fluorescent glucose analog. The data correspond to one representative experiment out of four independent replicates, each performed in triplicate. *, P< 0.05; **, P< 0.01; ***, P< 0.001.