Figure 1: Identification of Arid5b as a candidate transcriptional co-regulator for Sox9.

(a) Effect of Sox9 on Col2a1 expression in NIH-3T3 and C3H10T1/2 cells. NIH-3T3 (left) and C3H10T1/2 cells (right) were infected with control (Cont) or Sox9 adenovirus at 20 MOI and cultured for 4 days. Total RNA was isolated and subjected to real-time PCR. Data are shown as the mean±s.d. (n=3) *P<0.01 (versus Cont); Student’s t-test. (b) NIH-3T3 (left) and C3H10T1/2 cells (right) were infected with control (Cont) or HA-Sox9 adenovirus and cultured for 4 days. Cell lysates were analysed using immunoblotting with anti-HA and anti-β-actin antibodies. Sox9 expression was similar in NIH-3T3 and C3H10T1/2 cells. (c) Expression of Arid5b mRNA in NIH-3T3 and C3H10T1/2 cells. Total RNA was isolated from 80% confluent NIH-3T3 and C3H10T1/2 cells and analysed by real-time PCR. Data are shown as the mean±s.d. (n=3). *P<0.01 (versus NIH-3T3); Student’s t-test. (d) Expression of endogenous Arid5b in NIH-3T3 and C3H10T1/2 cells. Cell lysates were isolated from NIH-3T3 and C3H10T1/2 cells and analysed using immunoblotting with an anti-Arid5b antibody. β-actin was used as a loading control. Bands representing both the longer and shorter splice variants of Arid5b are visible. (e) Expression of Arid5b in developing limb buds. The expression of Arid5b, Col2a1 and Sox9 was similar in developing limbs in E12.5 mice, as determined by whole-mount in situ hybridization. Scale bar, 2 mm (upper panel), 1 mm (lower panel). (f) Immunohistochemical analysis of Arid5b in growth plate chondrocytes. Paraffin sections of a 3-week-old mouse tibia were subjected to H&E staining (upper panel) and immunocytochemical analyses using anti-Arid5b antibody (lower panel). Scale bar, 100 μm. H, hypertrophic chondrocytes; P, proliferating chondrocytes.