Fig 2: Gene expression o f p53 target genes after p53 loss.(A) Heat map generated by centroid linkage of the Euclidian distance of mean centered fold change values of transient transfection of shRNA plasmid 36 (T36) and stable line created with plasmid 36 (S36) using a control line (created with scrambled shRNA) as a common divisor, 2-((T36-Normalizers) – Control) compared to 2-((S36-Normalizers) – Control), (n = 1). The negative inverse used for fold changes between 0 and 1. Clusters were then divided into two categories, those with higher expression in T36 and those with higher expression in S36. (B) Fold change values plotted for genes involved in apoptosis or differentiation with a fold change between treated and control cells greater than two (C) Comparison of stable clones of varying p53 expression. Plot of the same gens as in (B) but between S36 and a stable line created with clone 34 (S34) show stable p53 knockdown irrespective of the level of knockdown, have a much more similar expression pattern than seen between transient and stable.

Fig 3: Confirmation of stable and transient knockdown of p53(A) Transcriptional activity of p53 is reduced with reduction in p53 levels Mean + S.E.M. of relative luciferase units (RLU) reported for stable (S) knockdown of p53 using shRNA plasmids and a transient knockdown using a mix of all three plasmids (T Mix) when compared to controls receiving scrambled shRNA sequences. (B) RT-PCR of p53 from a stable knockdown (S33), a transient knockdown (T33) and control (scrambled shRNA). (C) Immunoblot of p53 expression in a stable (S36) line and after transient knockdown of p53 (T36). The same blot was stripped and reprobed with anti-actin antibody. Similar results were obtained in three independent experiments and representative data are shown.