ing in Nucleus Pulposus Cells by Peptide 2.0 Inc. PD98059, U0126, and SB203580 were from Calbiochem. Human samples, isolation and culture of human nucleus pulposus cells Human tissue samples of lumbar pathological IVD were donated from consenting patients undergoing surgery for removal of herniated discs. The IVDs were maintained into DMEM:F12 and the nucleus pulposus was surgically dissected from the annulus fibrosus using a stereoscope. One tenth of each disk was acutely frozen in Trizol and the rest was used to establish primary 18289623 cell cultures. Briefly, nucleus pulposus cells were released after mincing with scalpels and an overnight treatment with 0.025% collagenase at 37C. Tissues were triturated with a pasteur pipette and debris removed with a 500 x g x1 min centrifugation; the resulting supernatant was washed and resuspended in DMEM:F12 1:1, supplemented with 10% FBS, 66M L-ascorbic acid, 1mM L-Glutamine, 1% Gentamicin, and 1% penicillin/streptomycin. After plating onto culture dishes, cells were grown at 37C and 5% CO2. Culturing media was replenished every 2-3 days and, when confluent, NP cells were trypsinized, counted, and split at a ratio of 1:3. Every 3-4 passages, some cells were frozen and stored in FBS:DMSO:glycerol:DMEM at a ratio of 50:10:10:30 at -90C or -180C, and were thawed with a similar success rate of 80% of the trials. Articular knee cartilages were donated from three patients with trauma and no history of joint disease and were cultured as previously described. Axiovert 200M inverted fluorescence microscope with a motorized stage and a Hamamatsu Orca-ER CCD camera; images from double or triple staining were obtained at the same focal planes of serial 0.5m thick Z dimension TMS optical sections and resulting Z-stacks were deconvolved with SlidebookTM. Immunoblotting, using total cell homogenates in RIPA buffer or concentrated, chondroitinase-treated culture media were performed as previously described. Twenty five g of protein or volumes corresponding to 500 g of cellular protein were analyzed 
per condition. Following separation with SDS polyacrylamide gel electrophoresis, proteins 21560248 were transferred to nitrocellulose membranes, and analyzed by indicated antibodies and the appropriate species specific secondary antibodies conjugated to horseradish peroxidase. Immunoreactivity was visualized by enhanced chemiluminescence. Chemiluminescence detection was linear at 30-60 sec of exposure for all antibodies used except for BC-3 which was 5 min. RT-PCR analysis Semi-quantitative PCR was performed as previously described. The oral consent was documented by an appropriate witness, following the protocol approved by the Ethics Committee, KAT Hospital, University of Athens. This protocol fully conformed to the ethical guidelines of the 1975 Declaration of Helsinki for Medical Research, including randomly assigned numbers to protect the anonymity of the patient. Immunocytochemistry, deconvolution microscopy, and Western blotting Cell fixation and immunofluorescence analysis was performed as previously described. For aggrecan detection, cells were treated postfixation with 5×10-5 U /L of Chondroitinase ABC for 3 hours at 37C. The primary antibodies were detected with species-specific rhodamine- or FITC-conjugated secondary antibodies as indicated in figure legends, the nuclear DNA with Hoechst 33258, and F-actin with Alexa Fluor 633phalloidin-. Amplification conditions were an initial denaturation step of 4 min at 94C followed b