Figure 2.

CDK5 downregulates p21 in TC cells. (A) Western blot analysis of p21 protein expression levels in BCPAP cells transfected with EV or a CDK5 overexpression plasmid. Cells were treated with 50 µg/ml CHX for 0, 2, 4 or 6 h to inhibit protein synthesis. Short and long exposures of p21 are shown. (B) Quantification of p21 protein levels. Data are presented as the fold change relative to time 0 for EV and CDK5 transfected cells. (C) Western blot analysis of p21 and CDK5 protein expression levels in BCPAP cells transfected with EV or CDK5, with or without treatment with 10 µM MG132 for 6 h. (D) Quantification of p21 protein expression. Data are presented as fold change relative to EV-transfected mock-treated cells. (E) Immunofluorescence staining of p21 (green) in BCPAP cells transfected with vector or CDK5, with or without MG132 treatment. The results indicate that CDK5 overexpression reduces p21 levels in TC cells and this effect was reversed following proteasome inhibition. Scale bar, 20 µm. (F) Co-immunoprecipitation analysis of CDK5 and p21 was performed to investigate the interaction between CDK5 and p21 in thyroid cancer cells. Panels show the interaction of CDK5 with WT p21 and MT p21 following proteasome inhibition using MG132. Red arrows indicate the presence of p21 in the immunoprecipitated complex. (G) Cell counting assay was performed to evaluate p21 WT and MT p21 on cell proliferation. For all blots, β-actin was used as the loading control. Values are presented as the mean ± SD from three independent experiments. Data were compared using an unpaired Student's t-test. *P<0.05, **P<0.01, ***P<0.001. CDK5, cyclin-dependent kinase; 5TC, thyroid cancer; EV, empty vector; HCX, cycloheximide; n.s., not significant; WT, wild-type; MT, S130A mutant.