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. 2019 Nov 21;19(1):109–123. doi: 10.1080/15384101.2019.1693748

Figure 5.

Figure 5.

High levels of mutant p53RR protein can partially compensate for the cooperativity defect. (a) mRNA expression analysis (RTqPCR) of p53 target genes in Trp53RR/RR embryos with indicated Mdm2 genotypes. Shown are expression values normalized to β-actin, normal n = 7, abnormal n = 4. (b) RTqPCR analysis of the expression of pro-apoptotic p53 target genes Bbc3 (Puma) and Bax in primary Trp53+/+, Trp53RR/RR and Trp53–/ – MEFs after 16 h treatment with 400 ng/ml doxorubicin. Shown are technical triplicates. In (a) and (b) * P < 0.05, ** P < 0.01, ns – not significant, error bars represent SD, two-tailed Mann Whitney test. (c) Quantification of apoptosis detected by flow cytometry with Annexin-V and non-permeable PI staining in Trp53+/+, Trp53RR/RR and Trp53–/– E1A-imortalized MEFs upon 16 h treatment with 10 μM Nutlin-3a, 400 ng/ml doxorubicin or a combination of both. (d) RTqPCR quantification of p53 target genes in E1A-MEFs from (c) upon treatment with Nutlin-3a and doxorubicin. Each dot indicates one biological replicate, ANOVA test. (e) Representative western blot of cytosolic, nuclear and mitochondrial fractions from primary MEFs (untreated and treated with 400 ng/ml doxorubicin), probed with anti-p53 antibodies. PCNA and Tom20 antibodies were used as fractionation controls.