Hat DO-1 reactivity should enhance substantially upon Nutlin treatment under the fixed circumstances employed in flow cytometry. Expectedly, flow cytometry quantitation shows that, even prior to Nutlin treatment, p53 ++ cells have considerably far more DO-1 reactivity than p53 — cells (Figure 1F). The functional importance of this `basal p53 activity’ will probably be investigated later within this report (Figure three). Interestingly, the p53 ++ cell population shifts to substantially greater DO-1 reactivity in the 1 hr time point, as predicted by epitope unmasking. A further raise is observed at 12 hr of Nutlin therapy, when total p53 levels have risen significantly as measured by Western blots (Figure 1C,F). Finally, given that GRO-seq is really a population average experiment, we performed immunofluorescence assays to test if our GRO-seq results could possibly be explained by huge p53 accumulation in just some outlier cells within the population in the 1 hr time point. On the other hand, these experiments discarded the notion of outlier cells: despite the fact that three cells show high p53 staining at the 1 hr time point, this quantity is just not drastically distinct than observed in control p53 ++ cells (Figure 1–figure supplement 1G,H). Altogether, these final results indicate that the low levels of p53 present in proliferating cancer cells suffice to straight activate a multifunctional transcriptional plan, which includes many canonical apoptotic genes, upon unmasking of the p53 transactivation domain by Nutlin. Having said that, as discussed later PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/21352867 within the paper (Figure 4), this conclusion doesn’t necessarily conflict with prior reports showingAllen et al. eLife 2014;three:e02200. DOI: 10.7554eLife.5 ofResearch articleGenes and chromosomes Human biology and medicineFigure 2. Worldwide analysis of p53 effects on RNA synthesis vs steady state levels. (A) MAplots for GRO-seq and GSK2256294A microarray gene profiling experiments in HCT116 p53 ++ cells after 1 hr and 12 hr of Nutlin therapy, respectively. Colors indicate regardless of whether genes scored as statistically diverse in both platforms (purple), inside the GRO-seq only (red) or the microarray experiment only (blue). (B) Couple of genes downregulated inside the microarray experiment show p53 binding inside 25 kb on the gene, suggestive of indirect regulation. (C) Bubble plots displaying relative signals derived from the GRO-seq and microarray experiments illustrate how genes with incredibly higher basal expression or really low transcription are certainly not drastically impacted in the steady state level as measured by microarray. For the CDC42BPG, KLHDC7A, ADAMTS7, LRP1 and ASTN2 loci, Figure 2. Continued on next pageAllen et al. eLife 2014;three:e02200. DOI: ten.7554eLife.6 ofResearch write-up Figure 2. ContinuedGenes and chromosomes Human biology and medicinethe signals had been replotted at 25-fold magnification. (D) Scatter plot showing comparative fold induction for p53 target genes transactivated at 1 hr Nutlin treatment amongst the GRO-seq and microarray experiments. (E) Q-RT-PCR indicates that a lot of low abundance transcripts upregulated by GRO-seq are certainly induced at the steady state level. (F) Box and whisker plots displaying the expression of several gene sets as detected by microarray. DOI: 10.7554eLife.02200.005 The following figure supplements are offered for figure 2: Figure supplement 1. Mechanisms of indirect gene repression by p53. DOI: 10.7554eLife.02200.006 Figure supplement 2. ChIP analysis of novel p53 target genes. DOI: 10.7554eLife.02200.differential timing of mRNA accumulation amongst arrest.