Activation of FOXO pathway and TXNIP induction. A: HepG2 and HuH-7 cells were treated with 250 μM MK-801 or NBQX for 12 h. cDNA was synthesized and real-time quantitative PCR was carried out using Taqman gene expression assay primers. Each reaction was performed in duplicate. The β-actin gene was used to normalize across assays and runs, and the threshold value (Ct) for each sample was used to determine gene expression levels. Each bar represents the mean ± SD of at least three replicates. B: HepG2 cells were treated with 250 μM MK-801 and expression of TXNIP and p27 was measured by real-time quantitative PCR for 72 h. Each bar represents the mean ± SD of at least three replicates. C: HepG2 cells were treated with 250 μM MK-801 for 0 to 96 h and Western blot analysis was performed using indicated antibodies. D: HepG2 cells were treated with 250 μM MK-801 for 0 to 60 min and Western blot analysis was carried out using indicated antibodies. The molecular weight of dephosphorylated band corresponded to Thr24 of FOXO1 E: FOXO1-pAcGFP-N1 plasmid was transfected to HepG2 cells and treated with or without 250 μM of MK-801. Nuclear translocation of FOXO1-GFP protein was observed with Olympus LX71 microscope.