Fig. 6.

The relationships between AMPK cascade and FOXO1 and LXRα transcription factors in the hydrogen peroxide-mediated downregulation of apoA-I gene. a Real-time RT-PCR. HepG2 cells (96 well plate) were transfected with 2.4 pmoles per well of scrambled siRNA (black columns), siRNA against FOXO1 (white columns), siRNA against LXRα (light gray columns or siRNA against AMPKa (dark gray columns) for 72 h before RNA isolation and treated with 250 μM of H₂O₂ for 24 h before RNA isolation. The activator of AMPK metformin (2 mM) was added 24 h before RNA isolation. Values are presented as means ± the standard error of the mean of four independent experiments. The statistical analyses of differences between compared groups were performed using a non-paired Student’s t test (**p < 0.05) and Dunnett’s criterion (#p < 0.05). b Western blotting analysis of subcellular localization of FOXO1 transcription factor. HepG2 cells were exposed to H₂O₂ (250 μM) for 2 h. JNK inhibitor SP600125 (10 μM) had been added 1 h before hydrogen peroxide. Crude cytoplasmic and nuclear fractions were isolated as described in Materials and methods and analyzed by Western assay. β-actin in cytosolic fractions was used as a normalization control. Diagram shows the quantification of Western blotting by densitometry. The Y-axis values correspond to the ratio (%) of nuclear pool of the FOXO1 transcription factor to its total amount. The total amounts of the transcription factor were normalized by β-actin. Values are presented as means ± the standard error of the mean of three independent blots. The statistical analyses of differences between compared groups were performed using a non-paired Student’s t test (**p < 0.05). c, d Real-time PCR calculation of ChIP. HepG2 cells were treated with hydrogen peroxide (250 μM) for 24 h. JNK inhibitor SP600125 (10 μM) had been added to cells 1 h before H₂O₂ administration. Histograms represent the relative level of FOXO1 (c) or LXRα (d) binding (100% in control cells) to apoA-I hepatic enhancer (HE) (black columns). Primers to GAPDH genomic DNA were used as non-specificity control (gray columns). AB against β-actin were used as the control for non-specific binding. Values are presented as means ± the standard error of the mean of three independent experiments. The statistical analyses of differences between compared groups were performed using a non-paired Student’s t test. There are no significant differences between levels of FOXO1 and LXRα binding to apoA-I HE in the control cells and in the cells under an oxidative stress