However, understanding more thoroughly how Sox9 mediates antagonism between cartilage and bone signaling pathways would have significant impact in understanding developmental and disease processes. In summary, this current study highlights a novel mechanism by which Sox9 acts as a transcriptional repressor of Spp1 to prevent matrix mineralization in heart valves and chondrocytes, in vitro. Despite these two connective tissue systems being functionally diverse, there is ever-increasing evidence to show that normal heart valve ECM shares molecular characteristics and signaling pathways with chondrogenesis . Similarly, pathological calcification of heart valve ECM has recently been referred to as a bone-like process, although parallel ��endochondral ossification�� events in valve interstitial cells have not been yet reported in human patients or mouse models of valve calcification. Nonetheless, our findings provide further support to show overlapping regulatory pathways between these two connective tissue systems and identify a previously unappreciated mechanism to prevent ossification in tissues that must remain cartilaginous for life-long function. Following culture and viral infection as described , one mouse valve explant per filter was mounted and von Kossa Everolimus mTOR inhibitor staining was performed as described previously . Chondrocyte cultures were stained following a similar protocol; cells were rinsed in PBS, fixed in 4% paraformaldehyde, washed 3 times in deionized water, and incubated in 5% silver nitrate solution for 1 hour under direct light in a reflective chamber. Chondrocytes were then washed in water, differentiated in 5% sodium thiosulfatepentahydrate for five minutes, and rinsed and counterstained for 20 minutes in 1% Alcian blue in 20% acetic acid. Quantification of von Kossa reactive area was performed using Image Pro Plus software and calculated as a percentage of von Kossa positive area over total area indicated by alcian blue staining. The induction of proinflammatory cytokines is a hallmark of renal inflammation and initiated by Adriamycin outside�Cin signaling, e.g. by activating Toll-like receptors that can convert a wide range of infectious and non-infectious stimuli into NF-kB signaling . Nuclear translocation of NF-kB induces cytokine mRNA transcription, protein translation as well as immediate secretion of the cytokine into the extracellular space .