Figure 3.

General workflow of the analysis of the effect of compression. A) Workflow scheme of the analysis of the effect of compression on gene expression and activation of signalling molecules in chondrocytes cultured in agarose. Immediately after their isolation from cartilage, chondrocytes are embedded in agarose gel. Cell/agarose constructs are cultured for 6 days to allow pericellular matrix deposition before being submitted to compression. At different time points after compression, total RNAs and proteins are isolated and analyzed by RT-PCR and Western blotting, respectively. B) Workflow scheme of the analysis of the effect of compression on gene promoter activity in chondrocytes cultured in agarose. After their isolation from cartilage, chondrocytes are first cultured in monolayer on plastic for one week, after which they are harvested and nucleofected with plasmids of interest. The transfected cells are embedded in agarose gels and the cell/agarose constructs are submitted to compression. After compression, the constructs are frozen in liquid nitrogen, lyophilized and lysed for reporter gene assay.