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. 2019 Apr 16;111(11):1202–1215. doi: 10.1093/jnci/djz051

Figure 6.

Figure 6.

Effect of sequestration of mutant TP53 by estrogen receptor-beta (ESR2) on mutant TP53-TP73 complex and TP73 activation. A) Schematic diagram comparing domains of TP53 and TP73, showing the N-terminal transactivation domain, DNA binding domain, oligomerization domain, and sterile alpha motif domain. Proteins are not aligned proportionately to the length of each domain. Numbers correspond to the amino acid residues that mark different domains. Note that the C-terminus of TP53 domain (aa 361–393) that binds to ESR2 is not conserved in TP73. B) Proximity ligation assay (PLA) for TP53-TP73 interaction was performed in MDA-MB-231 cells with or without knocking down ESR2 with ESR2-specific siRNA si-ESR2#1 for 48 hours. Scale bar = 20 μm. C) Co-IP of TP73 and TP53 was performed with MDA-MB-231 cells following ESR2 knockdown with si-ESR2 #1 for 48 hours. D) Quantitative immunoprecipitation for TP73 on CDKN1A and BBC3 gene promoters was performed in MDA-MB-231 cells following ESR2 knockdown with si-ESR2 #1 for 48 hours. E) Endogenous TP73 mRNA in MDA-MB-231 cells transfected with or without TP73 siRNA for 48 hours was analyzed by quantitative real time polymerase chain reaction (qRT-PCR). Bottom panel: TP73 protein levels in the cell lysates were analyzed by immunoblotting. F) TP53-target gene expression in MDA-MB-231 cells transfected with or without TP73 siRNA for 48 hours was determined by qRT-PCR. G) PLA for interaction between mutant TP53 and TP73 was performed in MDA-MB-468 cells with or without knocking down ESR2 with si-ESR2 #2 for 48 hours. Scale bar = 5 μm. H) TP53-target gene expression in MDA-MB-468 cells transfected with and without TP73 siRNA for 48 hours was determined by qRT-PCR. I) PLA for interaction between mutant TP53 and TP73 was performed in SK-BR-3 cells with or without knocking down ESR2 with si-ESR2 #2 for 48 hours. Scale bar = 5 μm. J) A model for the anti-tumorigenic role of ESR2 in the mutant TP53 context is shown. Showing two prototypic TP53-targets, p21 and PUMA, does not imply these are the only proteins participating in the ESR2-TP53 signaling crosstalk. All P values were determined by two-tailed Student t test. Error bars represent SD. IB = immunoblot.