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. 2019 Jul 16;10:3126. doi: 10.1038/s41467-019-11004-3

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© The Author(s) 2019, corrected publication 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — Fetal HNF4α-P2 isoform increase in patients with AH and its effect in HNF4α-P1. a Levels of bilirubin, INR and albumin levels in serum along ALD progression (values expressed as Mean ±SEM) and HNF4α footprint Z-Score. b Scheme of HNF4A gene fetal (P2) and adult (P1) isoforms structure and protein variants. c Real-Time quantitative PCR (qPCR) of HNF4A-P1 and P2 dependent isoforms, and lncRNA HNF4A-AS1 in the cohort of patients in Fig. 1. d Immunohistochemical detection of adult and fetal HNF4A protein variants in patients with AH (n = 9), and controls (n = 9), using N-terminal specific antibodies. e Semi-quantitative assessment of IHC signal for each antibody for nuclear staining. f–i HepaRG cells were retro-differentiated into tumor-derived Hepatocyte-like cells (HepaRG-tdHep); de-differentiation was induced with FBS and RNA was extracted at 4, 24, and 48 h (n = 3 for each time point); qPCR of g HNF4α-P1 and P2 isoforms, h phosphoenol-pyruvate carboxy-kinase (PCK1), clotting Factor VII (F7) and i vimentin (VIM) j, k HepG2 cells were transfected with plasmids encoding P1 (HNF4α2) and P2 (HNF4α8) variants. P1 was maintained at same dose while P2 was increased as indicated. RNA was extracted 12 h and 24 h after transfection (n = 3); qPCR of j HNF4α-P2 isoform and k PCK1. l–p HepG2 cells were transfected with siRNA targeting the first exon (1E) of HNF4α-P2 isoforms (n = 3), and RNA and protein was extracted at 48 h after transfection. qPCR of l HNF4α-P2 and m HNF4α-P1. n Western blot of HNF4α-P1 and HNF4α-P2 in nuclear extracts. qPCR of HNF4α-P1 targets related to o metabolic functions (PCK1, ALB and F7) and p bile acid synthesis and transport (BSEP, CYP7A1 and CYP27A1). q, r Primary human hepatocytes were silenced with siRNA-HNF4A-P2. q Supernatant was collected 48 h after transfection (n = 3 for each group) and total bile acids were quantified. r Glucose production in P2-silenced primary human hepatocytes. Significance was determined by unpaired, two-tailed Student’s t-test in a and c, by Fisher exact probability test in d, e and by two-tailed Mann–Whitney U test in g, i, l, m, o, p, q, r: *P < 0.05. For box-and-whisker plots: perimeters, 25th–75th percentile; midline, median; individual data points are represented