Figure 2

WT p53 downregulates Toca-1. (A) DU4475 cells were treated with indicated doses of CPT) for 24 h, and lysates subjected to IB with Toca-1, p53 and p21 antibodies. (B) Toca-1 mRNA levels were analyzed in DU4475 cells treated as above by qRT-PCR (2^-∆∆CT values for Toca-1 were normalized to GAPDH for each concentration of the drug, and graph depicts transcript levels relative to DMSO-treated cells (mean ± SD; triplicate samples)). (C) DU4475 cells were treated with CPT (4 μM) for the indicated times, and analyzed by IB as above. (D) DU4475 cells were treated with CPT (4 μM) for the indicated times were subjected to qRT-PCR analysis of Toca-1 mRNA levels as above. (E) DU4475 cells were treated with Nutlin-3 at indicated doses for 24 hours, and subjected to IB analysis as above. (F) p53-null HCC1806 cells were transfected with WT p53 expression plasmid (0 to 8 μg) for 24 to 48 hours, and levels of Toca-1, p53 and p21 were assessed by IB (β-actin served as a loading control). (G) HCC1806 cells were transfected with WT p53 plasmid, and after 24 hours with a siRNA against p53 prior to harvesting at 48 hours. Lysates were subjected to IB with Toca-1, p53 and β-actin antibodies. CPT, camptothecin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IB, immunoblot; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; SD, standard deviation; siRNA, small interfering RNA; Toca-1, transducer of Cdc42-mediated actin assembly; WT, wild-type.