Fig. 6

Exposure of mLO(-DAPT/D3) to fibrosis-associated cytokines. A Phase-contrast images after treatments of mLOs with (+ FIC) or without (Control) fibrosis-inducing cytokine cocktail (FIC, a mixture of TGFβ1, TNFα, IL-6, and IL-1β) for 5 days. Scale bar, 100 μm. B H&E staining showing a change in tissue architecture in mLOs after treatment with FIC. Note that cell platelike epithelial structures were destroyed by exposure to FIC. Scale bar, 100 μm. C, qRT-PCR analysis of changes in gene expression associated with hepatic epithelium (HNF4A, ALB, and SOX9), endothelium (PECAM1, CDH5, ICAM1, and SELE), and HSCs (PDGFRB, DESMIN, ACTA2, and COL1A1). Data are expressed as mean ± SD (n = 3, normalized to ACTB). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and NS (not significant) by unpaired t tests. D Whole-mount immunofluorescence images of mLOs for the expression of human collagen I and α-SMA, which are key markers of HSC activation. Scale bar, 200 μm. E Quantification of immunoreactive signals for human collagen I and α-SMA. Each black dot represents the percent positive area of immunoreactive signal in each mLO (n = 13 per group, **p < 0.01 and ****p < 0.0001 by unpaired t test). F Whole-mount immunofluorescence images of EC and hepatocyte marker (CD31 and HNF1β) expression in mLOs. Scale bar, 200 μm. G Quantification of immunoreactive signals for HNF4α and CD31. Each black dot represents the percent positive area of the immunoreactive signal in each mLO (n ≥ 4 per group, *p < 0.05 and **p < 0.0001 by unpaired t tests. H Single z-scan immunofluorescence images of E-cadherin expression in control and FIC-treated mLOs. Scale bar, 100 μm