After the last amplification cycle, PCR products were analyzed by melting curve ana lysis EPZ-5676 CAS in the Smart Cycler by slowly Inhibitors,Modulators,Libraries increasing the tem perature to 95 C. The reactions were run in triplicate with appropriate controls. The data were analyzed by using the Cepheid Smart Cycler soft ware and reported as threshold cycle. Change in gene expression was calculated as fold change 2, where . Statistical analysis for real time PCR Data are expressed as mean standard deviation. Statistical significance was assessed by the Student t test and P values Inhibitors,Modulators,Libraries 0. 05 were considered significant. GeneChip expression data from un stimu lated, rhOP 1 and OP 1AS treated chondrocytes maintained in high density monolayer culture were gen erated.

For the analysis of the expression data we used a three step analytical strategy, processing of raw inten sity values and normalization of profiles, examina tion of expression levels of gene categories that are relevant to articular cartilage, and comparison of gene expression Inhibitors,Modulators,Libraries changes between the two treatments OP 1AS to knockdown endogenous OP 1 expression vs. addition of exogenous rhOP 1. Analyzing the number of Inhibitors,Modulators,Libraries differentially expressed genes after rhOP 1 or OP 1AS, we found that rhOP 1 modulated expression of 4,057 genes, while OP 1AS treatment modulated expres sion of only 2,618 genes respectively. More genes were down regulated than up regulated by either treatment, rhOP 1 down regulated 3,365 genes vs 692 genes that were up regulated, while OP 1AS down regulated 2,364 genes and up regulated only 254 genes.

The functional groups of genes modulated by lack or excess of OP 1 are depicted in Figure 1. RhOP 1 primarily controlled genes responsible for molecular function, biological processes, and cellular components, while OP 1AS primarily affected genes controlling Inhibitors,Modulators,Libraries cellular processes and catalytic activity. Interestingly, either treatment up regulated fewer functional groups than the number that were down regu lated. For example, rhOP 1 induced only five functional groups vs four induced by OP 1AS, while rhOP 1 down regulated 19 functional groups vs 12 down regulated by OP 1AS. When the results were com pared between the two treatments, we found that very few gene groups with the same function were differen tially regulated by both treatments.

Groups regulated by both OP 1 conditions included the genes responsible find more information for cellular processes, development, protein binding, signal transducer activity and signal transduction. Analysis of catabolic genes, cytokines and their regulators Previously, we showed that OP 1 was able to counteract the catabolic activity of IL 1b and other catabolic mediators such as fragments of cartilage matrix, fibro nectin and hyaluronan. Therefore, it was of interest to determine the effects of OP 1 on genes regu lating pro catabolic activity.