Fig. 7: TGF-β signaling in SAH livers is a key determinant of hepatocyte identities.

a NicheNet-based cell interaction analysis summarizing (from left to right) 1. Key ligands that regulate hepatocyte identities in SAH livers, 2. Expression levels of ligands in different NPC cell types (Ligand producing “senders”) in SAH livers, 3. Log fold changes (LFC) in their expression levels in SAH livers compared to unaffected livers, and 4.NicheNet-based prediction of a ligand’s potential to regulate key hepatocyte target genes. TGF-β1 and HGF ligands showed the highest correlation to the gene set defining hepatocyte maturity. b Feature plot illustrating increased expression levels of TGF-β1 specifically in the NPC populations from SAH patients. c Western blot and RT-qPCR analyses showing ESRP2 reduction upon TGF-β1 treatment (5 ng/mL in DMEM/F-12 complete media for 36 h) and restoration upon addition of TGF-βR I/II inhibitor LY2109761 (50 μM for 48 h). TGF-β1 treatment increases steady-state mRNA levels of EMT-transcription factors Snai1 and Cdh2, which return to baseline after inhibitor addition. Phospho-Smad3 expression confirms TGF-β pathway activation, and its subsequent reduction upon inhibitor treatment confirms inhibitor efficacy. Data points reflect technical replicates (n = 3) for each condition, centers and error bars represent mean and Standard deviation, respectively. Presented data is representative of 3 independent trials. Pairwise Welch t-test was used to assess significance. *p < 0.05, **p < 0.01, ****p < 0.0001. d RT-PCR analysis of ESRP2 splicing targets (Slk, Slain2, and Tcf4) demonstrates significant exon skipping (93, 78, and 73 bp, respectively) following TGF-β treatment. These levels return to baseline upon the addition of the TGF-β inhibitor. Exon lengths are shown beside the gene name, and the differences in PSI values are shown as mean ± standard deviation below each image. e Schematic showing a proposed model of the combinatory roles of cytokines and Wnt signaling in regulating cellular transitions in hepatocytes after injury, which is severely misregulated in SAH due to misspliced downstream Wnt mediators. (Created in BioRender. Das, D. (2025) https://BioRender.com/3lchxkt).