Fig. 4

The combination treatment with metformin and abemaciclib changed the metabolome of cancer cells. a A heat map of differentially abundant metabolites obtained from the control, metformin (5 mM), abemaciclib (1.25 µM), and combination-treated groups at 48 h in the negative (ESI−) ion mode. b Pathway enrichment analysis of differentially abundant metabolites of all groups using MetaboAnalyst 4.0 website in the negative (ESI−) ion mode. c Chord plots generated using the Spearman rank correlations for the differentially abundant metabolites of the four groups in the negative (ESI−) ion mode. d Schematic of the altered metabolic pathways, including the TCA cycle, aspartate metabolism, and fatty acid oxidation, underlying the synergistic efficacy of metformin and abemaciclib. e–i Levels of the indicated metabolites in U251 cells treated with metformin (5 mM), abemaciclib (1.25 µM), or the combination for 48 h as determined by LC–MS/MS. Data are shown as the mean ± SD. *P < 0.01, **P < 0.001, ***P < 0.0001, and ****P < 0.00001 (two-way ANOVA; three technical replicates averaged in each)