Figure 4

p65 phosphorylation is induced by a p53-dependent death receptors expression. (a) Death receptor expression was measured in all cell lines by qPCR upon 8 h etoposide treatment (20 μM). The plot represents the relative quantification compared to control non-treated cells. (b) Fas receptor immunocytochemistry on D283-MED cells treated with 20 μM etoposide for 8 h. (c) Quantification by flow cytometry of Fas expression to the plasma membrane in D283-MED cells upon 8 h of etoposide treatment (20 μM). (d) D283-MED cells were transfected with 100 nM of siRNA directed against p53 or control-scrambled siRNA. After 48 h transfection, cells were treated with etoposide (20 μM, 8 h). Fas mRNA expression was assessed by qPCR. Results are expressed as fold levels induction compared with the control unstimulated cells transfected with scrambled siRNA. Knockdown of p53 was controlled by qPCR (not shown) and western blot (Figure 3c). (e) D283-MED cells were transfected with 100 nM siRNA directed against Fas or control-scrambled siRNA. After 48 h transfection, cells were treated with 20 μM etoposide for 6 h and phospho-p65 levels were measured by western blot. The plot represents the quantification of the blot shown. (f) D283-MED cells were treated with 20 μM etoposide in the presence or absence of the Fas antagonist ZB4 antibody (5 μg/ml) for 8 h. Cell viability was measured by MTS assay and expressed as % of control untreated cells. (g) D283-MED cells were treated for 6 h with etoposide (20 μM) in the presence or absence of the Fas antagonist ZB4 antibody. Phospho-Ser536-p65 levels were evaluated by western blot. The bands were quantified and the phospho-p65/p65 ratio was plotted for the different treatment conditions