Hat DO-1 reactivity need to enhance significantly upon Nutlin therapy below the fixed situations employed in flow cytometry. Expectedly, flow cytometry quantitation shows that, even just before Nutlin remedy, p53 ++ cells have substantially more DO-1 reactivity than p53 — cells (Figure 1F). The functional significance of this `basal p53 activity’ will probably be investigated later in this report (Figure three). Interestingly, the p53 ++ cell population shifts to substantially higher DO-1 reactivity in the 1 hr time point, as predicted by epitope unmasking. A additional raise is observed at 12 hr of Nutlin treatment, when total p53 levels have risen significantly as measured by Western blots (Figure 1C,F). Ultimately, considering that GRO-seq can be a population average experiment, we performed immunofluorescence assays to test if our GRO-seq results could possibly be explained by enormous p53 accumulation in just several outlier cells inside the population at the 1 hr time point. On the other hand, 
these experiments discarded the notion of outlier cells: although three cells show higher p53 staining in the 1 hr time point, this quantity will not be considerably different than observed in control p53 ++ cells (Figure 1–figure supplement 1G,H). Altogether, these outcomes indicate that the low levels of p53 present in proliferating cancer cells suffice to directly activate a multifunctional transcriptional plan, like lots of canonical apoptotic genes, upon unmasking from the p53 transactivation domain by Nutlin. Nevertheless, as discussed later PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/21352867 within the paper (Figure four), this conclusion doesn’t necessarily conflict with previous reports showingAllen et al. eLife 2014;3:KIN1408 supplier e02200. DOI: 10.7554eLife.5 ofResearch articleGenes and chromosomes Human biology and medicineFigure two. Worldwide evaluation of p53 effects on RNA synthesis vs steady state levels. (A) MAplots for GRO-seq and microarray gene profiling experiments in HCT116 p53 ++ cells after 1 hr and 12 hr of Nutlin treatment, respectively. Colors indicate whether genes scored as statistically different in both platforms (purple), inside the GRO-seq only (red) or the microarray experiment only (blue). (B) Handful of genes downregulated inside the microarray experiment show p53 binding within 25 kb of your gene, suggestive of indirect regulation. (C) Bubble plots displaying relative signals derived in the GRO-seq and microarray experiments illustrate how genes with pretty high basal expression or very low transcription are usually not considerably 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: 10.7554eLife.6 ofResearch short article 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 involving the GRO-seq and microarray experiments. (E) Q-RT-PCR indicates that a lot of low abundance transcripts upregulated by GRO-seq are indeed induced in the steady state level. (F) Box and whisker plots displaying the expression of various gene sets as detected by microarray. DOI: ten.7554eLife.02200.005 The following figure supplements are available for figure two: Figure supplement 1. Mechanisms of indirect gene repression by p53. DOI: ten.7554eLife.02200.006 Figure supplement 2. ChIP analysis of novel p53 target genes. DOI: 10.7554eLife.02200.differential timing of mRNA accumulation involving arrest.
