Figure 6

Microarray analyses identify the hypertrophic area as the main target of CNP treatment. E15.5 tibiae were isolated, incubated with or without CNP (1 μM) and DMSO or SB202190 (10 μM) and micro-dissected into the resting/proliferating, hypertrophic, and mineralized regions prior to RNA extraction and microarray analyses. Analyses of microarray results from three independent trials using Genespring 7.2 (A) illustrated that the hypertrophic zone was most significantly responsive to CNP treatment, when compared to control conditions (B). Six times as many probe sets showed at least 2-fold expression changes in the hypertrophic zone when compared to either resting/proliferating or mineralized regions. Real-time PCR analyses on micro-dissected tibiae were used to validate selected microarray patterns. CNP induction of Ptgs2, the gene encoding cyclooxygenase-2, was confirmed (C). SB202190 treatment did reduce basal Cox2 mRNA levels, but did not interfere with CNP induction of Cox2. Tnfsf11, the gene encoding RANKL, was confirmed to be down-regulated in response to CNP treatment. Data represent means ± SD of three independent trials (p < 0.05).