ATM deficiency is associated with abnormal mitophagy, not increased mitochondrial biogenesis. (A) Expression of genes involved in mitochondrial biogenesis is intact in ATM-deficient and wild-type thymocytes. Nrf1, PGC1β, PRC, PGC1α, SOD2, and TFAM mRNAs were measured by reverse transcription real-time PCR analyses. mRNA levels were normalized using Actin mRNA as an internal control. N.S. indicates not statistically significant. (B-D) Accumulation of Parkin and impaired mitophagy in A-T human fibroblasts. (B) Parkin levels are elevated in mitochondria of A-T fibroblasts without induction of mitochondrial damage by CCCP. Normal or A-T human fibroblasts were treated with DMSO or 50μM CCCP for 4 hours, and equal number of cells per sample was submitted to fractionation into mitochondrial (M), cytoplasmic (C), and nuclear (N) subregions. Equal volume of the fractions was analyzed by Western blot for the expression of Parkin, COX-IV (mitochondrial marker), Topoisomerase I (Topo; nuclear marker), LC3 (autophagic marker), and lactate dehydrogenase (LDH; cytoplasmic marker). We also analyzed 20 μg of total (T) protein. The small amount of LC3 present at the nuclear fraction after CCCP treatment is probably because of a residual mitochondrial material that fractionated with the nuclear compartment because CCCP could potentially induce damage to the mitochondrial membrane and thus make these organelles more leaky. (C) Normal or A-T human fibroblasts were treated with DMSO or 50μM CCCP for 3, 16, or 24 hours, and proteins were isolated followed by immunoblotting against COX-IV, LC3 (autophagic marker), and Actin (loading control) in total cell lysate. (D) Normal or A-T human fibroblasts were treated with CCCP as in panel C followed by cellular fractionation as in panel B, and mitochondrial protein fractions were analyzed by immunoblotting against COX-IV and LC3. Actin levels in the cytoplasmic fractions of these samples also were analyzed to verify equal loading based on cell number.