Blocking the non-canonical Wnt pathway can inhibit the specification of WB-F344 cells into myfibroblasts.

<p>(a) mRNA expression of Wnt 5b. (b) Immunostaining of α-SMA and F-actin. (c) The fluorescent intensity of α-SMA. (d) The fluorescent intensity of F-actin. (e) α-SMA was determined by qRT-PCR. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

The non-canonical Wnt signaling pathway is involved in HPCs expansion and differentiation.

<p>(a) β-catenin immunostaining (×200). (b) Expressions of β-catenin, frizzled1, frizzled4 and frizzled5, representing the canonical Wnt signaling, were measured by qRT-PCR. (c) Expression levels of Wnt5b and Wnt5a, representing the non-canonical Wnt pathway, were measured by qRT-PCR. (d) Expression levels of receptors, frizzled2, frizzled3 and frizzled6, were measured by qPCR. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

2-AAF advanced the progress of cirrhosis.

<p>(a) Sirius Red Staining (100×) and immunohistochemistry of desmin, α-SMA, Col I and Col IV (×200). (b) The relative expression levels of Col I and Col IV were measured by qRT-PCR. (c) The relative expression levels of α-SMA and TGF-β1 were measured by qRT-PCR. (d) Western blot of α-SMA. (e) The hydroxyproline content of liver tissue. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

Non-canonical Wnt signaling pathway was activated during the specification of WB-F344 cells into myfibroblasts.

<p>(a) Immunostaining of α-SMA and F-actin. (b) The fluorescent intensity of α-SMA and F-actin. (c) Expression levels of α-SMA, Col I and Col IV. (d) Wnt5a, Wnt5b, frizzled2 and β-catenin were determined by qRT-PCR. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

2-AAF promoted the activation and expansion of HPCs, and promoted the differentiation of HPCs to myofibroblasts.

<p>(a) Immunohistochemistry of CK19 and OV6 (×200). (b) The change in the OV6-positive labeling area. (c) The relative expression level of CK19 was measured by qRT-PCR. (d) Double immune-staining of α-SMA (red), OV6 (green) and DAPI (blue), merged α-SMA, OV6 and DAPI (×200). (e) Serial section immunostaining of α-SMA, CK19 and desmin in the 2-AAF(H) group (×200). *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p