Fig. 2.

Runx2 target gene identification. To identify mitotic target genes of Runx2, we applied a functional genomics strategy. Genes were selected that exhibit alterations in steady-state mRNA during progress from mitosis into G₁, that are sensitive to Runx2 siRNA, and that have promoters with Runx consensus motifs. The first two criteria were assessed by cDNA array-based gene profiling (SuperArray Bioscience Corporation), and the final criterion was assessed through a bioinformatics analysis by using TFSEARCH (48). (A) Mitotic cells were released into G₁, and RNA was taken at 0, 1.5, 3, and 6 h for analysis. (B) siRNA knockdown of Runx2 was monitored by Western blot analysis at concentrations of 50, 100, and 200 nM. Fluorophore-conjugated siRNA oligonucleotides were transfected in parallel to determine transfection efficiency. Micrographs show localization of siRNA oligonucleotides in cells with ≈95% efficiency at 100 nM. (C) Venn diagram indicates the number of genes in each of the three functional groups. Thirty-one target genes satisfying all three criteria were analyzed by hierarchical clustering based on cell cycle expression data (D) and expression in the Runx2-knockdown experiment (E) (see SI Data Set 1 for primer information). Color maps are applied to standardized gene expression data: pure blue, −3; pure white, 0; and pure red, 3.