Figure 1: 5-HT6R is highly expressed in bone and its activation attenuated in vitro osteoblast differentiation in primary calvarial osteoblasts.
(A) After total RNA was isolated from brain and bone in female 10-week-old ICR mice, the expression levels of serotonin receptors were analyzed by real-time PCR and normalized to that of β-actin. (B) Calvarial defects were created in female 10-week-old ICR mice. Arrowhead indicates the defect region (upper). The mRNA level of 5-HT6R at 1 day and 3 weeks was analyzed by real-time PCR and normalized to that of β-actin (bottom). (C,D) After primary calvarial osteoblasts from 1-day-old ICR mice were cultured with osteogenic supplement medium (OS) for 0, 1, 3, and 7 days, the mRNA level analyzed by RT-PCR (C) or real-time PCR (D) and was normalized to that of β-actin. (E–G) Cells were treated with OS in the presence of 1 and 10 μM ST1936 for 5 or 14 days. Differentiation was assessed by ALP staining (E), and Alizarin Red staining (F). The intensity of Alizarin Red staining was determined by optical density (G). (H,I) ALP activity was analyzed in the presence of the indicated concentrations of ST1936 and 1 μM SB258585 for 5 days (H) or analyzed in the presence of 10 μM ST1936 for 5 days, 24 h after transfection with 5-HT6R-siRNA (I). Data shown represent the means ± SEM of three independent experiments. *p < 0.05, vs. control. #p < 0.05, vs. OS. &, p < 0.05, vs. 10 μM SB258585 or vs. 5-HT6R siRNA with 10 μM ST1936.
