Compound 1 causes increase in pyruvate kinase (PK) activity. (A) Schematic representation of the metabolic changes observed in Snu398 cells upon LDHA inhibition indicating a significant rise in fructose-1,6-bisphosphate that is known to potentiate PK activity. (B) Compound 1 causes dose-dependent increase in PK activity in Snu398, but not HepG2 cells. Cells were incubated with dimethyl sulfoxide (DMSO) or increasing doses (Snu398 cells, closed circles) or 10 μM (HepG2 cells, open circles) of Compound 1 for 6 h and whole-cell extracts were isolated and subjected to PK activity analysis. (C) Compound 1 causes dose-dependent increase in Pyruvate kinase M2 (PKM2) tetramerization in Snu398 cells. Snu398 cells were incubated with DMSO control or increasing doses of Compound 1 for 6 h and whole-cell extracts were isolated and subjected to native gel electrophoresis followed by western immunoblotting with PKM2-specific antibody. NAD+, nicotinamide adenine dinucleotide; TCA, tricarboxylic acid cycle.