Endoplasmic reticulum stress actively suppresses hepatic molecular identity in damaged liver (2020)
Figure 6

Figure 6. ERS contributes to LIVER-ID gene downregulation and loss of hepatic identity in septic mice. (A) Similar analysis as in Fig.1C using transcriptomic data from the liver of sepsisBIM mice (16h after intraperitoneal injection of live E. coli).

(B-C) Enrichment plots from GSEA performed using LIVER-ID genes (B) or the response to ERS gene set (GO:0034976) (C) as the gene set and transcriptomic differences in sepsisBIM vs Control mouse liver as the ranked gene list.

(D) Comparison of the transcriptome of the indicated liver injury models (details are provided in Appendix Fig.S20 and Table EV6) with that of the developing mouse liver performed as described in the Materials and Methods. PC1 is the first principal component which represents 63.55% of the variability within the mouse liver differentiation study. PC1 was used to project the liver injury studies. APAP, Acetaminophen overdose - Model of drug-induced acute liver injury. CCL4, Carbon tetrachloride hepatotoxicity - Model of drug-induced chronic liver injury. MCD-HF, Methionine-choline deficient diet with high fat - Model of NASH/fibrosis.

(E) Box plots showing Log2 FC for ERS UP, DOWN or unchanged genes in sepsisBIM vs Control mouse liver (6 mice per group). Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-way ANOVA with Welch's correction and Dunnett's Modified Tukey-Kramer pairwise multiple comparison test was used to assess statistical significance, *P < 0.05.

(F) RT-qPCR analyses of selected ERS UP genes and LIVER-ID TFs monitoring expression changes in the liver of sepsisBIM vs Control mice (6 mice per group). The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC sepsisBIM/Control is statistically different from 0, *P < 0.05.

(G) Nuclear extracts from livers of control or sepsisBIM mice were subjected to Western blot or Simple Western immunoassay with antibodies against HNF4A, NR1H4 or FOXA2. LMNA was used as loading control.

(H) Nuclear extracts from livers of sepsisBIM mice pre-treated for 4 consecutive days with vehicle or 500 mpk TUDCA were subjected to Western blot with antibodies against HNF4A, NR1H4, FOXA2 or XBP1S. TFIIB was used as loading control. Additional mice are shown in Appendix Fig.S22D.

(I) Densitometric quantification of the protein expression data from 6 mice precondition shown in panel (H) (3 mice per condition) and Appendix Fig.S22D (3 additional independent mice per condition). The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC TUDCA/Vehicle is statistically different from 0, *P < 0.05.

(J) Serum alanine aminotransferase (ALT) (left) and aspartate aminotransferase (AST) activities (right) from sepsisBIM mice pre-treated for 4 consecutive days with vehicle or 500 mpk TUDCA (10 mice per group). The bar graphs show means ± SD (standard deviations). Student's t-test was used to assess statistical significance, *P < 0.05.