Fig. 3: Metformin promotes lysosomal degradation of the DHFR protein.

A qRT-PCR and western blot analyses of DHFR expression in HepG2 cells under metformin (2.5 mM) treatment for the indicated hours. B Western blot analysis of DHFR expression in response to cycloheximide (CHX, 1 µg/mL) with or without metformin (2.5 mM) treatment in HepG2 cells for the indicated hours. C Western blot analysis of Flag expression in response to cycloheximide (CHX, 1 µg/mL) in HepG2 cells expressing 3XFlag-DHFR with or without metformin (2.5 mM) treatment. D HepG2 cells were treated with PBS, MG132 (5 μM) or 3-MA (1 mM) with or without metformin (2.5 mM) for 12 h. DHFR protein levels were determined by western blot analysis. E HepG2 cells were pretreated with PBS or 3-MA (1 mM) for 6 h and then treated with CHX (1 µg/mL), metformin (2.5 mM) or both for 12 h. DHFR protein levels in the indicated cells were then determined by western blot analysis. F HepG2 cells overexpressing 3XFlag-DHFR were pretreated with PBS or 3-MA (1 mM) for 6 h and were then treated with CHX (1 µg/mL), metformin (2.5 mM) or both for 12 h. Flag protein levels in the indicated cells were then determined by western blot analysis. G PLC cells were treated with or without metformin (2.5 mM) for 24 h, and the medium was then replaced with LysoTracker staining buffer for 2 h. Then the cells were subjected to immunofluorescence assay. DHFR (green), lysosomes (red) and nucleus (blue) were visualised by confocal fluorescence microscopy. Scale bars, 10 µm. H Western blot analysis of the expression of autophagy-associated genes and DHFR in HepG2 cells treated with metformin (2.5 mM) for 12 h. Actin was used as the loading control. I Western blot analysis of the expression of Beclin1 and DHFR in HepG2 cells overexpressing 3XFlag-EV or 3XFlag-Beclin1. J Western blot analysis of the expression of Beclin1 and DHFR in HepG2 cells expressing NTC shRNA or shRNA against Beclin1 (shBeclin1) with or without metformin (2.5 mM) treatment for 12 h. Band intensities for protein expressions in the western blot assay were quantitated by ImageJ and normalised to Actin. Data are presented as the mean (±SEM) of three independent experiments. Statistical significance was assessed by ANOVA followed by Dunnett’s multiple comparisons test. *** indicates P < 0.001 compared between the indicated groups. ‘ns’ indicates no significant difference between the indicated groups.