Supplementary Materials, Methods and Figures from MCT1 Inhibitor AZD3965 Increases Mitochondrial Metabolism, Facilitating Combination Therapy and Noninvasive Magnetic Resonance Spectroscopy

Abstract

<p>This file contains Supplementary Materials, Methods and Figures relating to the manuscript as follows: Supplementary Materials: Antibodies for western blotting, Supplementary Methods: LDH activity assay, ATP measurements, confocal microscopy, histopathology analysis of tumors, high resolution NMR data acquisition and processing. Supplementary Figures S1-S7: Figure S1 shows time series spectra of changes in hyperpolarized 13C-lactate signal over time following addition of hyperpolarized 13C-pyruvate in control and AZD3965-treated Raji human lymphoma cells. Figure S2 shows that AZD3965 has no effect on LDH activity in Raji cells. Figure S3 shows confirmation of increased ATP levels with AZD3965 treatment in Raji and Hut78 human lymphoma cells as shown by a bioluminescent assay. Figure S4 shows that AZD3965 treatment has no effect on functioning mitochondrial density as revealed by confocal microscopy images of Mito Tracker Red staining n Raji cells. Figure S5 shows increased potency of AZD3965 (cell counts and % cell death) following co-treatment with the mitochondrial complex I inhibitor metformin in Kelly and IMR-32 human neuroblastoma cells (harbouring different levels of MCT4 expression) that is associated with increased mitochondrial metabolism (at least in Kelly cells). Figure S6 shows increased potency of AZD3965 (cell counts and % cell death) in Hut78 human lymphoma cells following co-treatment with either metformin or the mitochondrial pyruvate carrier UK5099. Figure S7 shows histopathology data of tumor necrosis and perfusion indicating that these parameters were comparable in the two tumor size groups used in the in vivo spectroscopy and growth inhibition experiments.</p>