Nell-1 primary response gene

We hypothesized that Nell-1, as a growth factor, is supposed to stimulate some molecular mediators, which transmit signals to downstream and finally affect chondrogenic differentiation. The molecular mediators which respond to Nell-1 in short time are our research objects, we called them primary response genes referred to pervious report . To find out Nell-1 primary response gene candidates, we first used microarray assays to analyze the differences of gene expression profile between rhNell-1 protein (100 ng/ml) and control (PBS) treatment. For screening real primary response genes, which is not regulated by new synthesis protein, ATDC5 cells were subjected to serum starvation, followed by protein synthesis inhibitor CHX (10 μg/ml) treatment for 30 min and then treated with PBS (control) or rhNell-1 protein (100 ng/ml) for another 30 min. Cell total RNA samples were sent to the UCLA DNA Microarray Centre (Los Angeles, CA) where target preparation and hybridization to the Affymetrix Murine 430 2.0 GeneChip was carried out following the standard protocol (http://www.affymetrix.com). This GeneChip contains over 39,000 full-length mouse genes and EST clusters from the UniGene database. The data obtained from the hybridization was preprocessed using Affymetrix GeneChip Command Console Software (AGCC) and Expression Console Software to generate probe set intensity data (Affymetrix, Santa Clara, CA). Expression values were further filtered by retaining only those probe sets with a fold change of at least 1.5 in rhNell-1 protein treatment samples compared with controls.