Fig. 1.

Novel Wnt agonists are inducers of β-cell proliferation. (A) Chemical structures of 5-thiophene pyrimidines. (B) Dose-dependent effects of 1a on β-cell proliferation. Relative luminescence unit (RLU) was measured by CellTiter-Glo assay after 7-day incubation of growth-arrested R7T1 β cells with 1a, which was added at day 0 and refreshed at day 4; experiments were performed in quadruplicate. All data in this paper are presented as mean ± SD, unless otherwise specified. (C) Dose-dependent effects of 1a on the activation of Super (8X) TOPFlash reporter. HEK293 cells transfected with Super (8X) TOPFlash reporter were treated with 1a at the indicated concentrations 24 h after transfection. Luciferase activity was measured 48 h after compound treatment. (D and E) The proliferative effect of 1a on rat primary β cells. (D) After incubation for 72 h with 2 μM 1a, replicating β cells were identified by double-immunofluorescence staining by using anti-C-peptide antibody to mark β cells (green) and antibody against Ki-67, a proliferation marker (red). Nuclear DNA was stained with DAPI (blue). (Scale bar, 10 μm.) (E) The percentage of Ki-67-positive cells of all C-peptide-positive cells, which corresponds to the fraction of replicating β cells. The number of proliferating primary β cells increased after incubation for 72 h in the presence of 2 μM 1a compared with DMSO control: 1a at 2 μM increases the replicating β cells ≈2-fold. (F) Compound 1a stimulates the translocation of β-catenin into the nucleus. Nuclear β-catenin accumulation (green arrows) was increased in 10 μM 1a-treated HEK293 cells; in DMSO-treated control cells, β-catenin staining is weak and appears to concentrate at the cell surface. (G) 1a-stimulated β-cell proliferation was abolished by the Wnt signaling antagonist Sulindac. R7T1 cells were treated with 10 μM 1a, 60 μM Sulindac, or 1 μM 2a as indicated in the figure. CellTiter-Glo activity was measured 7 days after compound treatment.