Figure 2

Mianserin suppresses Rspo2-induced activation of Wnt/β-catenin signaling in chondrogenic cell lines. (A,B) HCS-2/8 human chondrosarcoma cells transfected with the TOPFlash reporter plasmid to quantify Wnt/β-catenin signaling activity were treated with either 120 ng/ml recombinant human Rspo2 (rhRspo2) and 20 ng/ml recombinant human Wnt3a (rhWnt3a) (A) or 90 ng/ml rhWnt3a alone (B) along with increasing concentrations of mianserin for 24 h. Firefly luciferase activity of the TOPFlash reporter was normalized to the TK promoter-driven Renilla luciferase activity (n = 5). (C,D) ATDC5 cells were cultured with 1% ITS for 2 weeks to induce chondrogenic differentiation and subsequently treated with either 200 ng/ml rhRspo2 (C) or 90 ng/ml rhWnt3a (D) for 48 h in the presence of the indicated concentrations of mianserin. After staining with Alcian blue, proteoglycans in the cell lysates were quantified by measuring optical densities at 620 nm (n = 4). (E–H) Chondrogenically differentiated ATDC5 cells were treated with 200 ng/ml rhRspo2 for 48 h in the presence of the indicated concentrations of mianserin. Expression levels of each mRNA were quantified by quantitative RT-PCR, and were normalized for those of Gapdh and also for untreated cells (n = 4). Mean and SD are indicated. *P < 0.05 and **P < 0.01 by one-way ANOVA followed by Tukey’s post-hoc test.