Figure 2

PDK4 downregulation is observed in mesenchymal cells, and mediates metabolic rewiring. (A) A schematic representation of the metabolic rewiring observed in TGFβ-induced mesenchymal cells. Red highlight indicates increased flux from glucose to the TCA cycle and then to glutamate in the mesenchymal cells. (B) Heatmap based on microarray gene expression data showing the mRNA levels of PDK1-PDK4 in the parental cells (P) and mesenchymal derivatives (M). (C) Quantitative RT-PCR showing PDK4 mRNA levels in parental and mesenchymal cells. In B and C, data shown is the average of three separate biological samples generated at the same time. (D) Western blots with the mitochondria showing the PDK4 protein levels in parental and mesenchymal derivatives. VDAC was used as the protein loading control. The red asterisk denotes the upper band that specifically corresponds to PDK4. (E) Immunoblotting for PDK4 and EMT markers in A549 parental (Par) and resistant cells. The resistant clones were generated by culturing A549 cells in the presence of 0.5 μM GDC-0973 and 0.2 μM GDC-0941 for 2 months. Data from two independent clones is shown. (F, G) HCC827 parental and mesenchymal cells were infected with lentivirus expressing RFP or PDK4 with a GFP reporter. The GFP-positive cells were selected by FACS, cultured in growth media containing ¹³C-U-glucose overnight, and then subjected to LC-MS analysis. (F) Glucose to TCA cycle/glutamate contributions were plotted based on the percentages of (M + 2) citrate, (M + 2) malate and (M + 2) glutamate in each metabolite’s respective total pool. (G) Pyruvate to glutamate contribution was plotted based on the ratio of glucose → glutamate (the percentage of (M + 2) glutamate in total glutamate pool) over glucose → pyruvate (the percentage of (M + 3) pyruvate in total pyruvate pool). Pyruvate to citrate or malate contribution was similarly plotted. Data are plotted as mean ± SEM. **p < 0.01.