Figure 3: Molecular analyses of the annulus fibrosus (AF) cells.

(a) Quantitative real-time reverse transcription PCR (qRT–PCR) analyses of the expression of ligament-related genes in Mkx+/+ and Mkx−/− mice. The mRNA levels of ligament markers in Mkx+/+ mice were normalized to 1. Error bars represent s.e.m. *P<0.05, **P<0.01, ***P<0.001. Statistical differences were assessed with Student’s t-test. (b) qRT–PCR analyses of ligament- and cartilage-related gene expression in human AF cells transfected with a small interfering RNA for MKX (siMKX-a and siMKX-b) or the negative control (siNega). The mRNA levels of ligament markers in human AF (HAF) primary cultured cells transfected with siNega were normalized to 1. Error bars represent s.e.m. *P<0.05, **P<0.01, ***P<0.001. Statistical differences were assessed with Student’s t-test. (c) Immunohistochemistry (IHC) of the intervertebral disc (IVD) of 10-week-old Mkx+/+ and Mkx−/− mice. IHC shows little decreased Bgn and Col14 protein expression in Mkx−/− mice compared with Mkx+/+ mice. Red: Bgn and Col14. Blue: DAPI. Scale bars, 100 μm. (d) Western blot analyses of human AF cells. Western blotting showed decreased BGN and increased SOX9 protein expression following siMKX induction of HAF cells.