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. 2015 Aug;185(8):2194–2205. doi: 10.1016/j.ajpath.2015.04.021

Figure 3.

Figure 3

Wnt5a decreases β-catenin expression and activity and cell proliferation in liver cells. A: Treatment (48 hours) with 3 μg/mL human recombinant Wnt5a significantly decreases the TopFlash activity in human hepatoblastoma cell line (HepG2)–treated cells compared with phosphate-buffered saline (PBS). Wnt5a has no effect. B: Wnt5a-conditioned media (CM) derived from L cells show a dose-dependent decrease in HepG2 cell thymidine incorporation compared with treatment with media collected from control L cells. C: Treatment of transiently transfected HepG2 cells with 3 μg/mL recombinant human Wnt5a or HepG2 cells, transiently transfected with Frizzled-2–green fluorescent protein (GFP) expression plasmid, shows significantly lower TopFlash activity than HepG2 cells transiently transfected with pcDNA plasmid and treated with PBS. A more pronounced and synergistic decrease in TopFlash activity is seen with the treatment of Wnt5a on HepG2 cells transiently transfected with Frizzled-2–GFP. The transient transfection of Frizzled-2–GFP expression plasmid was measured by immunofluorescence (IF) for GFP that shows approximately 30% of cells to be transfected at 24 hours after transfection. D: Treatment of pcDNA-transfected HepG2 cells with 3 μg/mL recombinant human Wnt5a or transient expression of Frizzled-2–GFP in HepG2 cells leads to a significant decrease in thymidine incorporation at 48 hours when compared with pcDNA-transfected HepG2 cells treated with PBS. Combining the treatments elicits a more pronounced and synergistic effect. E: Addition of growth factors (GFs; epidermal growth factor and hepatocyte growth factor) to mouse growth medium (MGM) increases thymidine incorporation in primary mouse hepatocytes after 48 hours in culture. The addition of Wnt5a to MGM or growth factor media significantly decreases cell proliferation after 48 hours. F: Representative WB using whole cell lysates from hepatocytes cultured in MGM with Wnt5a for 48 hours shows a notable decrease in levels of β-catenin, cyclin-D1, and Frizzled-4 expression, and notable increases in Frizzled-2 and Ror2 when compared with media alone. No changes were observed in total glycogen synthetase kinase (GSK)-3β protein. Equal protein loading was verified by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). ∗P < 0.05, ∗∗P < 0.01. Original magnification, ×200 (C).