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.
