Figure 3. SPIN1 reduces p53 stability by enhancing MDM2-mediated ubiquitination.

(A) and (B) p53-half-life is increased by SPIN1 knockdown. (A) HCT116^p53+/+ cells transfected with scramble or SPIN1 siRNA for 48 hr, were treated with 100 μg/ml of cycloheximide (CHX), and harvested at different time points as indicated. The p53 protein was detected by WB analysis, quantified by densitometry and plotted against time to determine p53-half-lives (B). (C) and (D) SPIN1 overexpression shortens the half-life of p53. HCT116^p53+/+ cells transfected with pcDNA or Flag-SPIN1 for 48 hr were treated with 100 μg/ml of cycloheximide and harvested at indicated time points for WB analysis with indicated antibodies (C). The intensity of each band was quantified, and normalized with β-actin and plotted (D). (E) SPIN1 promotes MDM2-induced p53 ubiquitination. HCT116^p53-/- cells were transfected with combinations of plasmids encoding HA-MDM2, p53, His-Ub or Myc-SPIN1, and treated with MG132 for 6 hr before being harvested for in vivo ubiquitination assay. Bound and input proteins were detected by WB analysis with indicated antibodies. (F) SPIN1 enhances MDM2-mediated p53 proteasomal degradation. HCT116^p53+/+ cells were transfected with plasmids encoding HA-MDM2 and Flag-SPIN1, and treated with MG132 for 6 hr before harvested, followed by WB analysis with antibodies as indicated. (G) Ectopic SPIN1 does not change p53 protein level without MDM2. MEF^{p53-/-; Mdm2-/-} cells were transfected with combinations of plasmids encoding p53 with or without Flag-SPIN1, followed by WB analysis using antibodies as indicated.