Figure 3. Reciprocal regulation between FOXM1/NF-κB and MAT1A.

A) Protein levels of MATα1, FOXM1, p50, p65, HNF4, AFP, CYP2E1, and ACTIN were measured using western blotting in primary mouse hepatocytes at the time of isolation (0 hour = 0h) and up to 6 hours (6h) after plating. B) Protein levels of MATα1, FOXM1, p50 and p65 after overexpressing wild type MAT1A (1A OV), catalytic mutant of MAT1A (1Am) or siRNA knockdown (1A si) as compared to empty vector (EV) or scramble (SC) controls, respectively, in HepG2 cells. Numbers below the blots are densitometric values, expressed as percent of EV or SC in mean ± SEM from three experiments. *p < 0.05 vs. EV or SC; #p<0.05 vs. 1Am. C-D) Protein levels of MATα1, FOXM1, p50, and p65 after FOXM1 expression (FOXM1 OV) or siRNA knockdown (FOXM1 si) in HepG2 (C) and SAMe-D cells, which were also co-transfected with EV or MAT1A (D). Numbers below the blots are densitometric values, expressed as percent of EV or SC. Results are expressed as mean percent of control ± SEM from three experiments. *p < 0.05 vs. respective controls. E) Effect of FOXM1 siRNA or FDI-6 on NF-κB-driven promoter activity after 24h treatment in HepG2, SAMe-D cells, and human hepatocytes. *p < 0.05 FDI-6 vs. DMSO or FOXM1 si vs. SC. F) EMSA was performed by using a 32-bp double-stranded synthetic DNA containing two NF-κB motifs of the FOXM1 promoter and recombinant MATα1, p50, p65, FOXM1, PHB1 (all 100ng) alone or combined. The probe only served as a negative control. Results represent a total of at least 3 independent experiments.