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Figure 3 | BMC Biotechnology

Figure 3

From: Flow cytometry-based functional selection of RNA interference triggers for efficient epi-allelic analysis of therapeutic targets

Figure 3

Epi-allelic hypomorphic analysis to determine the threshold of p53-dependent responses to ionizing radiation. (a) Non-linear correlation between p53 and p21 mRNA levels in p53 epi-allelic primary human endothelial cells. Graded p53 knockdown in human umbilical cord endothelial cells (HUVEC) with shp53-3, shp53-10 shp53-13 or negative control (shLuc) (left panel) corresponded to a non-linear reduction in p21 gene expression (right panel). Data represent the mean ± SD of three (p53) and five (p21) measurements representative for two independent experiments relative to control cells (set as 100%). Statistical analysis was carried out using ANOVA followed by Tukey’s post hoc tests, *P < 0,05. (b) Non-linear correlation between p53 and p21 mRNA levels in p53 epi-allelic primary human endothelial cells after irradiation. Irradiation of p53-epi-allelic HUVEC with 2 or 8 Gy did not affect p53 mRNA levels but increased p21 mRNA expression in a non-linear, p53-dependent manner. The data represents the mean of duplicate samples ± range. (c) Ionizing radiation induced H2AX phosphorylation in p53 epi-allelic primary human endothelial cells. H2AX phosphorylation, a measure of DNA double strand break induction, in HUVEC, correlates with greater p53 knockdown. The data represents the mean of duplicate samples ± range. (d) Cell proliferation in p53 epi-allelic primary human endothelial cells with shRNA against p53 after irradiation. The number of EdU positive p53-epi-allelic HUVEC following ionizing radiation, as a measure of cell division, increases with p53 knockdown efficiency. The data represents the mean of duplicate samples ± range. (e) Dose response relationship between p53-levels and radiation-induced p21 activation by epi-allelic analysis. Ionizing radiation-induced p21 activation displays a sigmoidal dose-dependency on p53 gene expression.

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