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. Author manuscript; available in PMC: 2015 Nov 1.
Published in final edited form as: Hepatology. 2014 Sep 10;60(5):1753–1766. doi: 10.1002/hep.27285

Fig. 1.

Fig. 1

Establishing a model of sustained fibrotic liver in zebrafish. (A–A”’) In [Tg(fabp10a:CFP-NTR)gt1; Tg(Tp1:mCherry)jh11; Tg(hand2:EGFP)pd24] larvae treated with DMSO at 5 days-post-fertilization (dpf), quiescent HSCs showed a star-like configuration (A’, inset), whereas ECM protein laminin was almost undetectable (A”) and NRCs distributed among hepatocytes (A, A”’). (B–B”’) Hepatocytes were ablated by MTZ and allowed to regenerate for 50 hours. In the MTZ-treated regenerating livers, the number of HSCs increased (B’) with a rare deposition of laminin (B”). A population of cells co-expressing hepatocyte-specific CFP and NRC-specific nuclear mCherry was observed throughout the MTZ-treated regenerating livers (B”’, inset, white arrows) while bright red NRCs remained CFP negative (B”’, inset, yellow arrowheads). (C–C”’) The larvae were pretreated with 1.5% EtOH from 2.5 to 3.5 dpf and then concurrently treated with MTZ from 3.5 to 4.5 dpf and allowed to regenerate for 50 hours. HSCs increased in number and lost complex cytoplasmic processes (C’, inset). Elevated laminin deposition was observed in the EtOH/MTZ-treated regenerating livers (C”, inset). A population of cells co-expressing hepatocyte-specific CFP and NRC-specific nuclear mCherry was appreciated (C”’, inset, white arrows) while bright red NRCs remained CFP negative (C”’, inset, yellow arrowhead). (D) Representative western blot showed upregulation of laminin protein in the EtOH/MTZ-treated regenerating livers. Numbers show relative expression of laminin protein after normalization against β-actin protein. n=300 dissected larval livers per condition in three experiments. (E) Percentage (mean±SD) of CFP+ cells in the population of Tg(Tp1: mCherry)-positive cells. 85.5±1.6% of Tg(Tp1:mCherry)-positive cells were CFP+ in the MTZ-treated regenerating livers, while 18.7±1.8% of Tg(Tp1:mCherry)-positive cells expressed CFP in the EtOH/MTZ-treated regenerating livers. Cells in 5 planes of confocal images from 5 individual larvae were counted. Asterisks indicate statistical significance: ***p<0.001. All confocal images are single-plane images except A’, B’ and C’, which are projection images (n=30 larvae per condition in three experiments). Scale bar, 20µm. EtOH, ethanol; SD, standard deviation.