Fig. 3

MicroRNA-27b overexpression alters metabolite levels while suppressing PDH complex activity. a MCF7 and 4175 cells were plated and transiently transfected with the indicated oligonucleotides. After 24 h the overlying growth medium was collected and analyzed to determine lactate concentration. b Lactate assays were repeated in stably transfected cells in the same manner as in panel a. Additionally, lysates of the underlying cells were collected and the intracellular lactate (c). These data were normalized to the cell protein level determined by BCA assay. Lysates were also collected for pyruvate (d) and citrate (e) assays. Seahorse mitochondrial stress test quantitating OCR and ECAR in ZR75 (f and h) and 4175 cells (g and i) in response to miR-27b expression levels. Results are normalized to BCA protein of the cells in each well which were collected after each assay (j) Cell lysates were probed for global PDH complex activity in the samples a PDH complex activity assay kit. All PDH activity and extracellular metabolite data are normalized to the total cell protein in each well by BCA assay. Data from intracellular metabolite assays (lactate, pyruvate and citrate) was normalized to the cell number counted immediately prior to collection