Figure 3. Upon Oxidative Stress p53 is Stabilized and Accumulates in Mitochondria.

A, B. Robust (A) and rapid (B) stabilization of wt p53 in cells treated with H₂O_2. Doses and times as indicated. Immunoblot, PCNA loading control.

C. Endogenous p53 accumulates in a punctate perinuclear pattern that co-localizes with CypD. C6 rat glioma cells (wt p53) were left untreated or treated with 0.4 mM H₂O₂ for 6 hrs. Immunofluorescence.

D. Rapid mitochondrial p53 accumulation upon oxidative stress. Cells were treated with H₂O₂ for 4 hrs. Mitochondrial fractions were prepared by sucrose gradient ultracentrifugation. PCNA serves as indicator of purity of mitochondrial fractions and as loading control for crude and cytosolic fractions. CypD serves as mitochondrial marker and loading control for mitochondrial fractions.

E. Upon oxidative stress, p53 accumulates in the mitochondrial matrix. Isolated mitochondria from HCT116 p53 +/+ cells treated with 0.5 mM H₂O₂ for 4 hrs shown in (D) were further subjected to submitochondrial fractionation. M, crude mitochondrial; OM, outer membrane; IMS, intermembrane space; IM, inner membrane; MA, matrix.

See also Figure S3.