Knock-down of Sp1 reveals multiple targets in the p53 signalling pathway are altered. Sp1 knockdown was achieved using Ambion siRNA. RNA was extracted 48 and 72 hours post transfection, labelled and hybridised to human U133 plus 2.0 arrays. Analysis of microarray data was carried out using GCOS, Array assist and the DAVID online analysis tool. Statistical significance was determined using an unpaired two-tailed t test, summarised in the workflow shown in Fig 5A. Knockdown was confirmed by QPCR using TaqMan® gene expression assays (Fig 5B). Due to variability in knockdown produced by transient transfections, the biological replicates are shown as individual bars. The gene list identified using PLIER was sorted for statistically significant (p > 0.05) gene expression changes of greater than 1.2 fold. Subsequent analysis of this list using DAVID identified a number of changes occurring in the p53/P21 regulatory pathway (panel C; adapted from DAVID [[39, 40], and http://www.genome.jp/kegg/). Stars denote genes which were significantly upregulated (red) or down-regulated (black). Validation of a subset of genes was carried out using Applied Biosystems TaqMan® gene expression assays, according to manufacturer's instructions. QPCR expression data for Bid, p21, serpine, P53AIP and Sp1 are shown in Fig 5, panel B for HCT116 cells following Sp1 knockdown harvested 48 (panel Di) or 72 (panel Dii) hours post-transfection. The biological replicates are shown as individual bars; designated n1 and n2.