Fig 7. Human P53 associates with M0069tochondrial Sirt3 and modulates its level through Proteasome Pathway.

(A, B) p53 regulates Sirt3 level. Sirt3 expression at RNA (A) and protein level (B) were measured in p53 wild type (A549 and HCT116 p53+/+) and p53 deficient cells (H1299 and HCT116 p53-/-). Data are presented relative Sirt3 mRNA in p53 containing cells. (C, D) P53 interacts with SIRT3 in-vivo. HCT116 p53-/- cells were transfected with FLAG-p53 and Myc-Sirt3 alone or in combination. Twenty-four hours post-transfection, cells were harvested in immunoprecipitation assay buffer (IP Buffer). Cell lysates were immunoprecipitated with anti-FLAG followed by immunoblotting with anti-Myc. Five percent of whole-cell extract (WCE) was used as an input and was directly immunoblotted with respective antibodies. (D) Interaction of endogenous P53 and SIRT3. HCT116 p53+/+ cells were stimulated with Triptolide (50 nM) for 6 hours. Cells were harvested in IP buffer and cell lysates were immunopreipiatated with P53 (DO1) antibody followed by immunoblotting with SIRT3 antibody. Five percent WCE was directly immunoblotted with SIRT3 and P53 antibody. (E) SIRT3 turnover in p53+/+ and p53-/- HCT116 cells. Cells were incubated with 50 μg/ml of Cycloheximide for the indicated time. Cell lysates were subjected for immunoblotting with SIRT3 (above panel), p53 (Middle Panel) and β-actin (lower panel). (F,G) SIRT3 degradation is through Proteasome Pathway involving SKP2 E3-ligase. HCT116 p53-/- cells were pretreated with MG132 (2 μM) for 1 hour and then were treated with Cycloheximide (50 μg/ml) for indicated time. SIRT3 and β-actin levels were detected by immunoblotting with respective antibodies. Indicated cell lysates were immunoblotted with anti-SKP2 to examine the expression of SKP2 level (G). (H) TL alters SKP2 levels in p53-/- cells. HCT116 (p53+/+ and p53-/-) cells were stimulated with indicated dose of TL for 6 hours. Cell lsyates were immunoblotted with anti-SKP2, anti-SIRT3 and anti-beta Actin antibodies.