Figure 3

Mianserin reduces Rspo2-induced accumulation of β-catenin and phosphorylation of Lrp6 by suppressing binding of Rspo2 to Lgr5. (A–E) ATDC5 cells were cultured with 1% ITS for 2 weeks to induce chondrogenic differentiation and subsequently treated with 200 ng/ml rhRspo2 for 48 h in the presence of the indicated concentrations of mianserin. Expression levels of each mRNA by quantitative RT-PCR were normalized for those of Gapdh, and also for untreated cells. Mean and SD are indicated (n = 4). No statistical differences were observed by one-way ANOVA followed by Tukey’s post-hoc test. (F) Differentiated ATDC5 cells were cultured with 10 μM mianserin and 200 ng/ml rhRspo2 for 48 h. Representative Western blots and intensities of Lrp6 and β-catenin normalized for those of β-actin and also for untreated cells are shown. Mean and SD are indicated (n = 3). *P < 0.05 by one-way ANOVA followed by Tukey’s post-hoc test. (G) Differentiated ATDC5 cells were cultured with 10 μM mianserin and 200 ng/ml rhRspo2 for 1.5 h. Representative Western blots and intensities of phosphorylated Lrp6 normalized for those of total Lrp6 and also for untreated cells are shown. Mean and SD are indicated (n = 3). *P < 0.05 by one-way ANOVA followed by Tukey’s post-hoc test. (H) Cell surface binding assay of Rspo2-AP in the presence of increasing concentrations of mianserin on HEK293 cells expressing human Lgr5 or RNF43. Bound Rspo2-AP was normalized for the added Rspo2-AP. Mean and SD are indicated (n = 3). *P < 0.05 and **P < 0.01 compared to control cells expressing neither Lgr5 nor RNF43 by one-way ANOVA followed by Tukey’s post-hoc test.