Research Paper Volume 1, Issue 3 pp 335—349

Activation of p73 and induction of Noxa by DNA damage requires NF-kappa B

Figure 7. Dominant negative p73β blocks apoptosis induction and Noxa expression.. p73β wild type was introduced into p65 null cells and p73β dominant negative was introduced into p65 reconstituted cells by retroviral transfer. Then apoptosis induction and Noxa expression was assessed after treatment with 10 μM etoposide for 18 hr. (A) Floating and attached cells were then collected and stained with propidium iodide (PI). DNA content was analyzed by flow cytometry. Results are presented as percentage of cells with sub-G1 DNA content. The data shown represent the mean and SEM of three independent experiments. (B) S-100 extracts from p65 null (vector) and reconstituted cells (p65) were used to assess caspase activity by cleavage (arbitrary fluorescence units per minute [AFU/min]) of the fluorogenic substrate, Ac-DEVD-afc. The data shown represent the mean and SEM of three independent experiments. (C) Northern Blot for Noxa expression. Total RNA was extracted from p65 null (vector) and reconstituted (p65) cells after treatment with etoposide. Expression of Noxa was revealed by blotting with a specific radio-labeled probe. As loading control the ethidium bromide stained gel previous to transfer onto membrane is shown. This blot is representative of three independent experiments.