Fig 2: Induction of NFIL3 by acute ERS contributes to repression of genes involved in xenobiotic metabolism RT–qPCR analyses of Hlf, Tef, Dbp, and Nfil3 expression monitoring changes induced by acute ERS in MPH (3–5 independent experiments) (left panel) or mouse liver (five mice per group) (right panel). The bar graphs show means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine whether the mean Log2 FC ERS/control is statistically different from 0, *P < 0.05.Total protein extracts from MPH (left panel) or mouse liver (right panel) were subjected to Western blot with an antibody against NFIL3. ACTB was used as loading control. See Appendix Fig S7B for antibody validation.Chromatin fractions from MPH (upper panel) or mouse liver (lower panel) were subjected to Western blot with an antibody against NFIL3. Histone H3 was used as loading control.Enrichment scores from GSEA performed using LIVER-ID genes repressed by acute ERS in MPH (MPH ERS DOWN) as the gene set and liver transcriptomic changes induced by acute ERS and/or deletion of Nfil3 (NFIL3 KO) as the ranked gene lists were integrated and corrected for multiple testing using the BubbleGUM tool. For the NFIL3 KO ERS vs WT ERS comparison, the Core Enrichment genes (i.e., the subset of genes that contributes most to the enrichment result) were subjected to functional enrichment analyses using the ToppGene Suite. The top ranked KEGG Pathway with its Bonferroni-corrected P-value is shown.Box plots showing mRNA expression for 5 genes from the Core Enrichment from D involved in xenobiotic metabolism issued from the transcriptomic analyses. Shown are Log2 FC relative to the mean normalized expression in the WT control group (five 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. Two-way ANOVA with Bonferroni's post hoc test was used to assess statistical significance, *P < 0.05.The Integrated Genome Browser (IGB) was used to visualize ChIP-seq profiles for NFIL3 (green) and several LIVER-ID TFs (red) in the mouse liver at the Gsta3 gene locus. Levels of H3K27ac in MPH and cells from the non-parenchymal fraction (NPC) are shown in blue. The grey bar indicates the position of a BRD4 SE.

Fig 3: Summary of the molecular mechanisms involved in ERS-induced loss of hepatic molecular identity and implications for liver pathophysiology Model for ERS-mediated loss of hepatic identity. Initial loss in LIVER-ID TF activities involves reduced protein levels linked to PERK and SRC signaling ?, which is secondarily amplified by decommissioning of BRD4 SE ? and impaired feedback loops within the LIVER-ID TF hepatic network ?. This consequently leads to loss of hepatic molecular identity and partial dedifferentiation of hepatocytes ?. Induction of NFIL3 operates as an additional mechanism further contributing to active repression of the LIVER-ID TF network target genes, especially those involved in xenobiotic metabolism.Implications of the competitive equilibrium between the hepatic and ERS transcriptional programs in liver pathophysiology. Please also refer to the discussion.

Fig 4: Co-expression of STAT3 and NFIL3 correlated with chemotherapy resistance. (a) Western blot analysis of STAT3 protein levels in different generations of subline cells (above) and subsequent densitometric quantification changes in protein levels are represented as fold change comparison with JEG-3 cells (under). (b) The IC50 dose response curves of Mock, NC-treated and siNFIL3-treated in all subline cells tested in vitro. (c) Representative images of TUNEL-stained cells treated with shSTAT3 or NC control, and then exposed to chemotherapeutics for 24 hr. Blue DAPI-stained nuclei, green TUNEL-stained positive nuclei, scale bars 100 µm (left). The Respective TUNEL-positive cells are depicted as percent (right). (d) Cells were exposed to chemotherapeutics for 24 hr after Annexin V-FITC/PI staining flow cytometry analysis was performed. (e) p-STAT3, STAT3, NFIL3, Cleaved-Caspase 3, Caspase 3, and Bcl-2 protein levels in cells treated with chemotherapeutics for 24 hr. GAPDH was used as the internal control (above). Respective changes in protein levels are depicted as fold change (under). Representative data are shown from three independent experiments. **p < 0.01, ***p < 0.001

Fig 5: Enhanced IL-2/STAT5 signature and IL-2 sensitivity in activated Pik3cdE1020K/+ OT-1 T cells(A) GSEA for IL-2-STAT5 hallmark pathway.(B) Heatmap of log10 TPM + 1 values for genes (hierarchically clustered rows) defined as core enriched elements in (A). Green boxes, TFs; purple boxes, regulatory genes.(C) NFIL3, CD137, and BATF3 in peptide-stimulated OT-1 cells (n = 4, representative example).(D) CD25, BATF3, and NFIL3 in T cells from healthy controls and patients with APDS (on treatment with Sirolimus) stimulated with anti-CD3 plus anti-CD28 (n = 3, representative example).(E) CD25 on OT-1 cells stimulated with OVA257–264 or T4 (n = 4, representative example).(F) OT-1 cells stimulated with 10 nM OVA257–264 for 3 days, re-stimulated with IL-2, and evaluated for pSTAT5Y694 and pS6S240/244 (n = 3, representative example).(G) pS6S240/244 in OT-1 cells were stimulated with 10 nM OVA257–264 with or without vehicle control or anti-IL-2 and exogenous human IL-2 and then washed, rested, and re-stimulated with IL-2 (MFI in histogram; n = 4, representative example).See Figure S6 and Table S1.