p53-Induced Growth Arrest Is Regulated by the Mitochondrial SirT3 Deacetylase
Figure 5
Human BAG-2 associates with p53 and stabilizes the level of p53 in vivo.
(A) EJ-p53 cells were cultured to induce p53 expression upon withdrawal of tetracycline after 24 hours. As shown, input level of p53 and human BAG-2 are shown from total cell lysates obtained from the EJ-p53 cells in the presence (+tet) or withdrawal of tetracycline (-tet) from culture medium for EJ-p53 cells. Immunoblots were conducted with antisera against p53 and BAG-2 from 50 mg of total cell lysate. Bottom, shown is an immunoprecipitation conducted with anti -p53 followed by immunoblotting with anti –BAG-2 antibodies. (B) RNA interference (RNAi) of human BAG-2 was conducted with siRNAs (Ambion, sequence ID#137542) directed at endogenous human BAG-2 mRNA in the presence of p53 expression within EJ-p53 cells. Cells were maintained at 40% confluent growth in 5% CO2 and transfected with 7 mg of the 21nt siRNA in 30 mm Petri dishes. The control sample is representative of a transfection using scrambled 21nt RNA mixture provided by the manufacturer. In parallel cultures, both total cell RNA and protein was recovered and used to measure mRNA and protein levels by northern hybridization using cDNA probes for human BAG-2, HPRT, and p53 and immunoblotted with the antibodies corresponding to BAG-2, HPRT, and p53, respectively. (C) Thymidine incorporation into nascent genomic DNA was measured upon introduction of siRNA targeted against BAG-2 and scrambled RNAs in EJ-p53 cells induced for the expression of p53 after 24 hours. Values reflect the relative incorporation of [3H] thymidine versus DNA content. (D) Total cellular content of acetylated and total following induction of p53 in EJ-p53 cells and infection with the MSCV –hSirt3 retrovirus or transfection with small interfering RNAs (siRNA) against human BAG2 (BAG2 RNAi). Antibody against acetylated p53 as described was used to detect acetylated species of p53. (E) As shown in panel D, Human embryonic lung fibroblasts IMR-90 cells were infected with MSCV-SirT3 and the MSCV control vector. Sub-cellular mitochondria and nuclear fractions were isolated and collected. Immunoblots confirming the presence of pan-acetylated p53, Lamin B1 and adenine nucleotide translocase (ANT) are shown from each sub-cellular fraction.