Fig. 2.

Effect of CCN2 on the expression of VEGF in chondrocytes. (A: left panel) Expression of Vegf₁₆₄ gene in wild-type and Ccn2-deficient chondrocytes. Wild-type (open column) and Ccn2-deficient chondrocytes (closed column) were cultured until reaching confluent. Total RNA was collected and quantitative real-time RT-PCR analysis was performed using mouse homologue; Vegf₁₆₄ and Gapdh specific primers. Data are presented as mean and standard deviation of duplicate cultures. (A: right panel) Western blot analysis of VEGF protein in lysates of wild-type and Ccn2-deficient chondrocytes. Levels of VEGF are decreased in Ccn2-deficient chondrocytes. (B) HCS-2/8 cells were transfected with a Ccn2-expression plasmid (Ccn2), a Hif-1α-expression plasmid (Hif-1α), or empty vector (Cont). After 24 h, the cells were exposed to normoxic or hypoxic conditions for 24 h, and total RNA was isolated using ISOGEN reagent. Northern blotting for Vegf₁₆₅ mRNA expression and methylene blue staining for 28S ribosomal RNA were performed.