Fig. 2: Nrf2 is determinant for HPC activation.

a GSEA demonstrates up- and downregulated signaling pathways in undifferentiated HPCs as compared with differentiated cells. This panel shows the results of our analysis of microarray data from the publicly available GEO data set (accession number GSE7038), using Gene Ontology (GO) or Kyoto encyclopedia of genes and genomes (KEGG) enrichment. Signaling pathways were ranked based on normalized enrichment scores (NESs); positive and negative NESs indicate down- or upregulation, respectively, in undifferentiated HPCs. Specific pathways related to metabolic or redox pathways are highlighted in red and blue. b Area proportional venn diagram representing 84 common genes between the downregulated genes in transcriptomes originating from mature hepatocytes [GSE7038 and GSE28891] and NRF2 target genes (NCBI Entrez Gene Database, 4780). c Confocal microscopy was used to examine the nuclear localization of NRF2. Representative confocal images demonstrating nuclear (DAPI, blue) colocalization of NRF2 (green) in quiescent and activated BECs/HPCs; Z-stacks (1 micron) were taken and rotated in two dimensions. Differentiated cells clearly show no colocalization, whereas undifferentiated cells demonstrate peri-nuclear and nuclear colocalization. d Representative pictures of 10,000 cells acquired using the Amnis FlowSight cytofluorimeter, showing the bright field, nuclear stain (violet), NRF2 (yellow), and merge. The nuclear internalization is shown as percent of cells obtained by three independent experiments. e mRNA expression of Notch and Wnt pathway targets (Axin2, Myc, Sox9, Hes1, Hnf1a, Hnf4a, Hnf6), and hepatic maturation genes (albumin, CYP3A4, Ggt-1) in BECs/HPCs treated with a siRNA targeting NRF2 or a control siRNA (scrambled) for 24 h. Data in the graph are represented as mean ± SD. Statistical differences were assessed by student’s t test. *p < 0.05 vs scrambled; **p < 0.01 vs scrambled; ***p < 0.001 vs scrambled. f NRF2 binding to the HMOX1 and NQO1 promoters in primary hepatocytes and BECs/HPCs isolated from mice fed a control chow (L, lean), 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet (C, cholestasis), or methionine-choline deficient diet (S, steatohepatitis). PCR products were detected on 2% agarose gel. Band intensities were quantified with Image J software. Input DNA was used as a control. g Quantitative analysis of ChIP experiments. Fold increase was calculated over their respective primary hepatocytes from lean mice. Data in the graph are represented as mean ± SD of three experiments. Statistical differences were assessed by student’s t test. **p < 0.01 vs primary hepatocytes; ***p < 0.001 vs primary hepatocytes.