Tob1 expression in fetal, normal and osteoarthritic cartilage (PCR, immunostaining, in situ hybridization). (a) Conventional PCR demonstrates the expression of Tob1 in normal and (at a reduced level) in osteoarthritic cartilage samples (lanes 1 and 9, molecular weight standards; lanes 2–4, normal cartilages; lanes 5–7, osteoarthritic cartilages; lane 8, negative control). In all experiments the RNA was directly from the tissue (without isolation of cells before isolation of RNA). (b) Quantitative real-time PCR analysis for mRNA expression levels of Tob1 in normal (n = 10) and peripheral (pOA, n = 8) and central (cOA, n = 7) osteoarthritic cartilage as well as normal adult articular chondrocytes cultured with (n = 6) and without (n = 3) serum. Results are shown as ratios to glyceraldehyde-3-phosphate dehydrogenase. (c, d) Immunolocalization of Tob1 in human normal (c) and osteoarthritic (d) articular cartilage (in both the middle and upper deep zones of the cartilage are shown). (e) mRNA expression analysis of Tob1 in fetal growth plate cartilage of mice, with the use of in situ hybridization: detectable expression levels are restricted to the hypertrophic zone (and osteoblasts).