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. 2020 Oct 20;17(9):2415–2431. doi: 10.1080/15548627.2020.1827779

Figure 3.

Figure 3.

HNF4A upregulated Ulk1 expression through direct binding to the Ulk1 gene. (A) Differences between the means of expression levels at 5 and 10 week, and those at 20 week. TFs selected using the criterion having FDR < 0.15 were labeled and colored red or blue to indicate elevated or reduced expression, respectively. (B) TF expression that was significantly increased or decreased according to PCR array analysis was validated using qRT-PCR. Values represent mean ± SEM (n = 10). *** p < 0.001, compared to the LFD group. (C) ATF3 and HNF4A levels in the liver of human NAFLD patients were analyzed using TaqMan probe, compared to those from a person without fatty liver (Control, CON). (n = 5). (D) Hepatocytes were transfected with the indicated siRNA for 48 h; the target autophagy-related gene mRNA levels were measured using qRT-PCR. Values represent mean ± SD (n = 3). ** p < 0.01; *** p < 0.001, compared to the negative control. (E–F) Hnf4a-overexpressing hepatocytes were generated using an Hnf4a mouse retroviral vector. (E) The mRNA levels of Ulk1 and Ulk2 and (F) protein levels of ULK1 and LC3 were measured in Hnf4a-overexpressing cells. Values represent mean ± SD (n = 3). * p < 0.05; *** p < 0.001, compared to normal hepatocytes (control). (G) ChIP assay was performed using the HNF4A antibody in hepatocytes. *** p < 0.001, compared to the negative control (IgG). (H) The expression of autophagy-related genes in Hnf4a-overexpressing hepatocytes was analyzed using PCR array, and the difference in expression of autophagy-related genes is shown in the heatmap. (I) Genes that were upregulated based on PCR array analysis were validated using RT-PCR. ChIP: chromatin immunoprecipitation; FDR: false discovery rate; HFD: high-fat diet; IgG: immunoglobulin G; LFD: low-fat diet; mRNA: messenger RNA; NAFLD: nonalcoholic fatty liver disease; PCR: polymerase chain reaction; qRT-PCR: quantitative reverse transcription PCR; SD: standard deviation; SEM: standard error of mean; siRNA: small interfering RNA; TF: transcription factor