Effects of Nell-1 overexpression on mineralization and bone marker expression. (a) FRCC culture infected with 20 pfu/cell AdNell-1, stained with von Kossa stain. Control cell cultures were infected with Adβ-Gal. Experiments were performed in triplicate. Mineralized nodules are stained black. (b) Quantitation and statistical analysis of mineralized areas. AdNell-1–infected cultures demonstrated significantly greater mineralization than did Adβ-Gal controls. (c) AdNell-1–infected MC3T3 cells grown without ascorbic acid. Typical micronodule appearance is shown. Right panel represents alkaline phosphatase staining of a micronodule. (d–f) Microarrays of AdNell–infected MC3T3 cells on postinfection days 6, 9, and 12, respectively. Gene expression intensities have been normalized using standardized housekeeping genes (HKGs). Hybridization intensities of AdNell-1–infected cells are represented on the y axis. Hybridization intensities of Adβ-Gal–infected cells are represented on the x axis. HKGs r² represents the correlation of housekeeping genes (filled squares) between the two samples. ECMs r² represents the correlation of candidate gene expression (open squares) between the two samples. A photograph of the microarray reading is attached in the upper left corner of each diagram. A twofold or greater upregulation is represented in red, while a twofold or greater downregulation is represented in green (g) Table summarizing genes with a difference in expression that is twofold higher or lower after AdNell-1 infection. The ratio is calculated as Nell-1/β-Gal. Col, collagen.