Extended Data Fig. 4: Activation of PKM2 increases cellular OAA concentrations to inhibit LDH activity. Related to Fig. 4.
a. Relative whole cell OAA concentrations of RPMI 8226 cells treated with 40 μM DASA or vehicle overnight. Whole cell metabolites were extracted with 1:1 methanol:water containing 5 mM ninhydrin, with or without 12C OAA spike, derivatized, and analysed with GC-MS. b. Relative whole cell OAA concentrations of PKM2 and PKM1 expressing H1299 cells. c. Schematic of fractionation method. d. Relative mean signal intensity, determined by flow cytometry, of (left) ER-BODIPY red, (middle) Golgi tracker red, and (right) Mito tracker red stained H1299 cells, treated with 40 μM DASA or vehicle. e. Representative serial block face scanning electron microscopy (SBFSEM) image of an H1299 cell. Small organelles included in the small organelle fraction are colored blue. Representative golgi, endoplasmic reticulum (ER), and mitochondria are circled in white. Image is one slice from one representative cell. f. Whole cell volumes as well as fractionated volumes of 12 cells analysed using SBFSEM. g. Western blot of H1299 lysate and increasing concentrations of purified recombinant LDHA. Corresponding standard curve constructed from the average pixel density of each LDHA purified recombinant protein band and used to determine the amount of LDHA protein in H1299 lysate. h. In vitro LDHA activity assay, using purified recombinant LDHA and pyruvate and cytosolic OAA concentrations determined under 40 μM DASA or vehicle treatment in H1299 cells. LDHA activity was determined by analysing 13C1 lactate produced from 13C1 pyruvate by GC-MS. Western blot results are representative experiments of three independent replicates. Data is represented as the mean and error bars represent the standard deviation from n = 4 for (a), (b), (h), n = 3 for (d) of biologically independent replicates. Micrograph in (e) is a representative experiment of 12 biologically independent replicates. Standard curve in (g) is a representative experiment of 3 independent replicates in vitro. The western blot in (g) is a representative experiment of 3 biologically independent replicates. P values were determined by a two-tailed Student’s t-test.
