Figure 2

Inhibition of P53 expression by shRNA alters regulation of P53 target genes. (A) Protein (25 μg) from melanocytes, WS-1, Mel-RM and IgR3 cells that had been stably transduced with P53 shRNA or control shRNA was analysed for the expression of P53 by western blotting. The expression of GAPDH was determined to ensure equal loading. Arrowhead indicates expected molecular weight. (B) Relative quantification of BIRC5, CDC25C, PLK2 and SESN1 mRNA by real-time RT-PCR in melanocytes, Mel-RM and IgR3 cells that had been stably transduced with P53 shRNA or control shRNA. Results are shown as the relative normalised expression (target/β-Actin) of the target gene in cells transduced with control shRNA compared to cells transduced with P53 shRNA (2^-ΔΔCt). Values represent the mean ± SE. (C) Supervised hierarchical cluster analysis of 13 genes that were regulated by P53 in melanoma cells only and not in melanocytes. Genes are coloured according to their expression level, where up-regulated expression is represented by red, down-regulated expression is represented by blue, and equal expression is represented by yellow. (D) Supervised hierarchical cluster analysis of 16 genes that were regulated by P53 in melanocytes only and not in melanoma cells. Genes are coloured according to their expression level, where up-regulated expression is represented by red, down-regulated expression is represented by blue, and equal expression is represented by yellow.