1. Induction of cell death and AIF-induced large-scale DNA fragmentation is blocked by nuclear BNIP3.
Induction of cell death and AIF-induced large-scale DNA fragmentation is blocked by nuclear BNIP3. A, U251 parental, U251 shRNA nontargeting control, U251shRNABNIP3, and U251NLS-BNIP3 stable cells were treated with 2 mm TMZ for 48 h or with DMSO for control. U87 parental, U87NLS-BNIP3, and U87shRNABNIP3 stable cells were treated with 2.5 mm TMZ for 48 h. Percentage cell death was determined by acridine orange staining. B, U251 parental, U251shRNABNIP3, and U251NLS-BNIP3 stable cells were treated with TMZ as above, and genomic DNA was extracted and then run on a Bio-Rad pulse-field gel apparatus. The lower arrow indicates approximately where the 50 kb DNA fragments migrate. This experiment was repeated three times and results were quantified by densitometry. C, Mitochondrial fractions were isolated from U251 parental, U251shRNABNIP3, and U251NLS-BNIP3 that were untreated, or treated with TMZ as above. The lysates were Western blotted for AIF, HDAC1 (a nuclear protein), and cytochrome c oxidase II (a mitochondrial protein). D, Nuclear fractions were isolated from U251 parental, U251shRNABNIP3, and U251NLS-BNIP3 that were untreated, or treated with TMZ as above. The lysates were Western blotted for AIF, HDAC1, and cytochrome c oxidase. E, U251 parental, U251shRNABNIP3, and U251NLS-BNIP3 stable cells were treated with 100 ng/ml TMZ for 24 h. Relative caspase 3 activity was measured as outlined in Materials and Methods.
Teralee R. Burton et al. J. Neurosci. 2009;29:
©2009 by Society for Neuroscience