Fig. 1: Metformin sensitises hepatocarcinoma cells to MTX treatment by inhibiting DHFR.

A, B Cell numbers of parental (A) or MTX-resistant (B) HepG2 cells under treatment of different doses of MTX for 72 h with or without metformin (2.5 mM) were measured by a cell counting assay. The IC50 values of MTX in these cells were further calculated with Graphpad Prism 7.0. Data are presented as the mean (±SD) values. C Proteomic analysis of protein expression fold changes (FC) between MTX-resistant HepG2 cells and parental HepG2 cells and between MTX-resistant HepG2 cells treated with metformin (2.5 mM) for 48 h and MTX-resistant HepG2 cells treated with PBS. Proteins with significant alterations in MTX-resistant cells that were reversed by metformin are marked in black or red in the diagram. D Western blot analysis of DHFR expression in Parental HepG2 and MTX-resistant cells treated with MTX (15 nM), metformin (2.5 mM) or a combination of both for 48 h. E Western blot analysis of DHFR expression in HepG2, PLC and Hep3B cells treated with PBS or metformin (2.5 mM) for 48 h. F Diagram indicating DHFR’s essential role in purine and pyrimidine synthesis. DHFR catalyses the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). THF is further transformed into 10-formyl-THF by the folate cycle, which provides 1-C units for purine synthesis. DHFR also associates with thymidine synthase (TS) to generate dTMP from dUMP and 5,10-methylene-THF. G MTX-resistant HepG2 cells were treated with MTX (15 nM), metformin (2.5 mM) or a combination of both for 3 days, and the relative abundances of ATP and GTP were measured by LC-MS. H The cell cycle in MTX-resistant HepG2 cells treated with MTX (15 nM), metformin (2.5 mM) or a combination of both for 3 days was analysed by flow cytometry. I MTX-resistant HepG2 3XFlag-EV and 3XFlag-DHFR cells were treated with DMSO or a combination of MTX (15 nM), metformin (2.5 mM) for 3 days. Cell numbers in the indicated groups were measured by a cell counting assay. J MTX-resistant HepG2 cells were treated with MTX (15 nM), metformin (2.5 mM) or a combination of both with or without the addition of a 25 μM nucleotide mixture for 3 days. Cell numbers in the indicated groups were measured by a cell counting assay. K Flow cytometric analysis of the cell cycle in MTX-resistant HepG2 3XFlag-EV or 3XFlag-DHFR cells that were treated with DMSO or a combination of MTX (15 nM), metformin (2.5 mM) for 3 days. L Flow cytometric analysis of the cell cycle in MTX-resistant HepG2 cells treated with DMSO or a combination of MTX (15 nM), metformin (2.5 mM) metformin with or without the addition of a 25 μM nucleotide mixture for 3 days. Band intensities for protein expressions in the western blot assay were quantitated by ImageJ and normalised to Actin. Data are presented as the mean (±SD) values. Statistical significance was assessed by ANOVA followed by Tukey’s multiple comparisons test. *, **, and *** indicate P < 0.05, 0.01, and 0.001, respectively, compared between the indicated groups. ‘ns’ indicates no significant difference between the indicated groups.