Figure 2

Activated caspase assays for NP cell apoptosis. (a) Activated caspase-8 activity for all experimental conditions; there is marginally increased activity in NCCM + IF and further elevated activity for BCCM but no suppression of activated caspase-8 activity for any of the treatment groups. (b) Activated caspase-9 activity is upregulated in the 2% +IF group and markedly down-regulated by NCCM (P < .01) and also (although to a lesser extent) by BCCM (P < .01). (c) Levels of activated caspase-3 for 2%, 2% + IL-1β + FasL, NCCM + IL-1β + FasL and BCCM + IL-1β + FasL treated cells. Here the use of NCCM in the presence of IL-1β + FasL reduces activated caspase-3 activity to almost baseline - precisely as demonstrated using flow cytometry and AnnexinV and PI labeling. Interestingly, there is no protection from apoptosis using conditioned medium obtained from bovine NP cells (BCCM) indicating that anti-apoptotic signaling is uniquely conferred by NCCM (P < .01). The suppression of both activated caspase-3 and -9 by treatment with NCCM indicates an anti-apoptotic effect of NCCM upon NP cells in the presence of IL-1β + FasL that is not accounted for by the other treatment groups. RLU (depicted on the 'Y' axis) is the fluorometric activity that occurs in the presence of caspase activation that releases a substrate for luciferase that in turn is determined using fluorometric methods. In each case 2% NCCM was generated from pooled notochordal cells obtained from the IVDs of five non-chondrodystrophic canines (eight to nine discs/animal). Bovine NP cells were obtained from the caudal discs of five to six three-year old steers. The 2% BCCM was generated from a separate pool of bovine caudal discs similarly obtained, seeded within alginate beads and conditioned medium developed over three days. Abbreviations: IL-1β (Interleukin-1beta) FasL (Fas ligand), NCCM (notochordal cell conditioned medium), BCCM (Bovine Cell Conditioned Medium), NP cells (nucleus pulposus cells), RLU (Relative Luminescence Units).