Figure 7.

P53 is both necessary and sufficient for the induction of nSMase2. (a and b) MCF7 cells were seeded in 60 mm dishes and siRNA knockdown was performed using AS or p53 for 24 h. After that, vehicle or doxorubicin were added and cells were collected and immunoblotted for nSMase2, p53 and actin (a), or RNA was isolated and transformed to cDNA and quantitative real-time PCR (qRT-PCR) was performed for nSMase2 (b), *P<0.05. (c and d) MCF7 cells were seeded in 60 mm dishes and transfected either with control (pcDNA), increasing concentrations of WT p53 plasmid (c), or different p53 mutants (d). Cells were collected and RNA was isolated, after which it was transformed to cDNA. qRT-PCT was performed for nSMase2, *P<0.05 and **P<0.01. (e) MDA-MB-231 cells were treated with different concentrations of doxorubicin. Cells were collected, RNA was isolated and qRT-PCR performed for nSMase2. (f) MDA-MB-231 cells were transfected with AS or nSMase2 siRNA after which they were treated with vehicle or doxorubicin. Cells were collected and in vitro NSMase activity assay was performed