Research Paper Volume 1, Issue 3 pp 335—349

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

Figure 1. Apoptosis resistance in p65 null MEFs.. (A) retrovirus-mediated reconstitution of p65 null MEFs with p65 restores NF-κB function as measured by EMSA. Wild type (wt), p65 null (vector) and p65 null reconstituted MEFs were stimulated with 10 ng/ml TNFα for 6 hr. Nuclear proteins were extracted and equal amounts of extract incubated with a radio-labeled NF-κB consensus probe. (B) p65 null cells are resistant to genotoxin-induced apoptosis. Cells were treated with 10 μM etoposide or 5 mJ UV-irradiation for 18 hr. 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. **statistically significant by student t-test analysis (p<0.05). (C) S-100 extracts from p65 null (vector) and reconstituted cells (p65) treated with 10 μM etoposide 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.