Calreticulin expression stratified for treadmill-running and knockout.

<p>Representation of the <i>Calr</i> expression values of all individual samples plotted by genotype-group (Wild-type and <i>Dio2</i>^<i>-/-</i>) and treadmill-running-group (No Run and Run). Showing the significant reduction of <i>Calr</i> expression upon forced treadmill-running in wild-type-mice (<i>P</i> = 0,005) and the absence of change in the knockout-mice. P-values depicted are derived from Student T-Test.</p

Calreticulin expression in <i>DIO2</i> overexpressing hBMSCs.

<p>qPCR expression of <i>Calr</i> in hBMSCs transduced with either a control virus-vector (eGFP) or a <i>DIO2</i>-eGFP vector. Values of the RT-qPCR are displayed as the average±SEM, normalized for <i>GAPDH</i> expression and relative to the control sample at every timepoint. Differences were analyzed with Student T-Test ((*) P < 0.05).</p

Expression analyses in <i>Calr</i> overexpressing ATDC5 cells.

<p>RT-qPCR expression of <i>Calr</i>, <i>Dio2 and Acan</i> in ATDC5 cells transfected with either a <i>Calr</i>-containing vector for overexpression (<i>Calr</i>+) or 3 unique 27mer siRNA duplexes, specific for Calr, for knockdown (<i>Calr</i>-). Values of the RT-qPCR are displayed as the average±SEM, normalized for <i>GAPDH</i> expression and relative to the control samples (dashed line). Differences were analyzed with Student T-Test ((*) P < 0.05).</p

Overexpressing <i>Calr</i> resulted in significantly lower Alcian Blue (AB) staining intensities.

<p>Staining intensities of Alcian Blue staining measured with a photospectrometer (620 nm). Values are displayed as the average±SEM, relative to the control sample. Differences were analyzed with Student T-Test ((*) P < 0.05).</p

Genes Involved in the Osteoarthritis Process Identified through Genome Wide Expression Analysis in Articular Cartilage; the RAAK Study

<div><p>Objective</p><p>Identify gene expression profiles associated with OA processes in articular cartilage and determine pathways changing during the disease process.</p><p>Methods</p><p>Genome wide gene expression was determined in paired samples of OA affected and preserved cartilage of the same joint using microarray analysis for 33 patients of the RAAK study. Results were replicated in independent samples by RT-qPCR and immunohistochemistry. Profiles were analyzed with the online analysis tools DAVID and STRING to identify enrichment for specific pathways and protein-protein interactions.</p><p>Results</p><p>Among the 1717 genes that were significantly differently expressed between OA affected and preserved cartilage we found significant enrichment for genes involved in skeletal development (e.g. <i>TNFRSF11B</i> and <i>FRZB</i>). Also several inflammatory genes such as <i>CD55</i>, <i>PTGES</i> and <i>TNFAIP6</i>, previously identified in within-joint analyses as well as in analyses comparing preserved cartilage from OA affected joints versus healthy cartilage were among the top genes. Of note was the high up-regulation of <i>NGF</i> in OA cartilage. RT-qPCR confirmed differential expression for 18 out of 19 genes with expression changes of 2-fold or higher, and immunohistochemistry of selected genes showed a concordant change in protein expression. Most of these changes associated with OA severity (Mankin score) but were independent of joint-site or sex.</p><p>Conclusion</p><p>We provide further insights into the ongoing OA pathophysiological processes in cartilage, in particular into differences in macroscopically intact cartilage compared to OA affected cartilage, which seem relatively consistent and independent of sex or joint. We advocate that development of treatment could benefit by focusing on these similarities in gene expression changes and/or pathways.</p></div

Genes significantly differently expressed in OA affected cartilage.

<p>Shown are results of the microarray analysis (Discovery) and the replication for genes with at least 2-fold change (Dir.: direction of effects relative to OA; FC: fold change; Pval: P-value).</p

Representative slides of immunohistochemical staining.

<p>A) H&E staining. B) Toluidine blue staining. C) SERPINE1. D) CD55 (magnification 20x; insets show larger overview at magnification 4x; white scale bars indicate 50 µm and 200 µm, respectively). The left panels show preserved cartilage area (P) and the right panels show the OA affected cartilage area (OA).</p

Protein-protein interaction between the genes with expression changes of at least 2-fold (Table 1) as determined with STRING.

<p>Protein-protein interaction between the genes with expression changes of at least 2-fold (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103056#pone-0103056-t001" target="_blank">Table 1</a>) as determined with STRING.</p

Genes identified in robust genome wide approaches with fold-changes and P-values for OA versus preserved cartilage (OA vs P).

<p>(Ref: reference, where indicated gene was published as OA susceptibility gene; Pval: nominal P-value; FC: fold change; –: not detected on microarray).</p

Gene enrichment analysis in OA affected versus preserved cartilage.

<p>Analysis considering the biological processes option in DAVID (GOTERM_BP_FAT), the cellular compartment option (GOTERM_CC_FAT), or the molecular function option (GOTERM_MF_FAT) as indicated in the first column, using medium classification stringency for all genes significantly differently expressed between OA affected and preserved cartilage (GO-Term: GO-terms within the different clusters; Count: number of genes identified for the respective GO-term; Pct: percentage of genes from total number of genes tested; Enr.: fold enrichment of indicated pathway; Pval: P-value; Pval_adj: adjusted P-value; FDR: false discovery rate).</p