Figure 3: Effects of pterosin B on chondrocyte differentiation in vitro.

(a) Structure of pterosin B. (b) Primary chondrocytes prepared from Col11a2-CreER; Sik3^flox/flox mice were treated with vehicle, 0.5 μM 4-OH tamoxifen or 300 μM pterosin B for 5 days and subjected to western blot analysis. The images are representative of two independent experiments. (c) Real-time RT–PCR expression analysis of marker genes in pellet culture of mouse primary chondrocytes with pterosin B; n=3 pellets. (d) Metatarsal primordial cartilage from Sik3^+/+ mice were organ-cultured in 300 μM pterosin B or vehicle. Metatarsal primordial cartilage from conventional Sik3 knockout mice were used for the control. Semiserial histological sections were stained with safranin O-fast green-iron haematoxylin and immunostained for Col2 and Col10. The percentage of Col10-postive area per total length in the axial direction is indicated; n=3 metatarsals. (e) Primary chondrocytes were treated with 100 μg ml⁻¹ cycloheximide in the presence or absence of 300 μM pterosin B. Cells were lysed after treatment for 0, 24 and 36 h, and cell lysates were subjected to western blot analysis for Sik3 and Gapdh. The images are representative of two independent experiments. (f) Primary chondrocytes were incubated in the presence or absence of 1 μM MG132, 10 nM bafilomycin A1 or 300 μM pterosin B for 36 h. Cell lysates were subjected to western blot analysis for Sik3 and Gapdh. The images are representative of two independent experiments. Error bars denote the means±s.d. *P<0.05 and **P<0.01 by the Tukey–Kramer post hoc test. Scale bars, 100 μm.