Early chondrogenic differentiation is accompanied by NF-κB/p65 activation and transient Sox9 expression.
A: Nuclear translocation of the NF-κB subunit p65. Total extract (T), cytoplasmic (C) and nuclear (N) fractions were isolated at 0, 0.5, 1 and 2 hours in ATDC5 differentiation. Cytoplasmic marker: α-tubulin, nuclear marker: Histone H3. B: COX-2, iNOS, Il-6 and TNFα mRNA expression at 0–24 hours in differentiation (relative to t = 0 and corrected for β-actin). C: Sox9 mRNA expression during chondrogenic differentiation (left panel). Protein expression of Sox9, COX-2 and iNOS at 0–24 hours (middle panel) and for Sox9 also at 0, 7, 10 and 14 days in differentiation (right panels). Molecular weight markers (kDa) are depicted on the left of immunoblots and relative quantifications are depicted on top of immunoblots. * = p<0.05. D: mRNA and protein expression of Col2A1, Col10A1, RunX2 during ATDC5 differentiation E: Sections from 6 weeks old mouse growth plates (resting (RZ) and proliferative (PZ) zones) stained for p65, COX-2, iNOS and Sox9. Lower panels show appropriate negative controls. Bars = 50 µm.