Figure 7

Western blot analysis of cell cycle regulators. After 24 h incubation in a medium containing 2% fetal bovine serum (FBS), this medium was replaced with medium containing 0.5% FBS. Nucleus pulposus cells were cultured with no supplements for an additional 24 h (a). The cells were treated with 5 ng/mL transforming growth factor β1 (TGFβ1) for 24 h (b). At 2 h before the addition of TGFβ1, the cells were treated with 16 μM 10058-F4 (c), or with 30 μM PD98059 (d). The cells were harvested 24 h after the TGFβ1 treatment and lysed. Aliquots of the lysates were electrophoresed on 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein bands were blotted to a polyvinylidene diflouride (PVDF) membrane and probed with antibodies against c-Myc, p15, p21, and p27. β-Actin was used as a quantity loading control. Treatment with TGFβ1 without inhibitors (b) did not abolish c-Myc expression but decreased the level of cyclin-dependent kinase inhibitors (CKIs) (p21, p27) compared to the control, while treatments with inhibitors (c, d) diminished c-Myc and upregulated p21 and p27. In contrast, p15 levels were unchanged by any of these treatments.