Mutant p53 sequesters p73 in cancer cells.

<p>(<b>A</b>) Overexpressed HSP70 restores transcriptional activity of TAp63α isoform inhibited by p53 R175H mutant in H1299 cells (left diagram), while TAp73α remains inhibited (right diagram). All data points were carried out in triplicate by means of Dual Luciferase Reporter Assay. (<b>B</b>) Overexpressed HSP70 molecular chaperone releases TAp63α isoform from the complex with p53 R175H mutant in H1299 cells, whereas maintains TAp73α in this interaction. 24h post-transfection cells were lysed and p53 protein was immunoprecipitated with anti-p53 antibody (DO-1). The immunoprecipitated protein complexes were subjected to Western blot analysis. (<b>C</b>) H1299 cells were transfected with plasmids encoding p53 R175H and p63 or p73 and treated with MG132 (2 µM) overnight. Labelling for specific antibodies showed that under such conditions p73 coaggregates with mutant p53 in the nucleus, while p63 remains diffused in the same compartment. Analysis performed in the ImageJ software with the JACoP plugin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051426#pone.0051426-Bolte1" target="_blank">[88]</a> confirmed that the colocalization is high for p73 and p53 R175H, whereas p63 overlaps with mutant p53 only partially (diagram). Two colocalization factors were determined – Pearson’s coefficient (r) and Mander’s coefficients (M1 and M2) - using threshold value of 110 for images in p63 analysis (upper panels) and 95 for p73 (lower panels). (<b>D</b>) p63 and p73 protein solubility assay performed in H1299 cells cotransfected with plasmids encoding one of those two proteins parallel with p53 R175H and HSP70 revealed that p63 is absent in the most detergent resistant fraction, while a part of p73 pool remains aggregated and is present in the Laemmli sample buffer together with mutant p53 and HSP70. Detailed description of the method available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051426#pone.0051426.s001" target="_blank">Figures S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051426#pone.0051426.s002" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051426#pone.0051426.s003" target="_blank">S3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051426#pone.0051426.s004" target="_blank">S4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051426#pone.0051426.s005" target="_blank">S5</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051426#pone.0051426.s006" target="_blank">S6</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051426#pone.0051426.s007" target="_blank">S7</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051426#pone.0051426.s008" target="_blank">S8</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051426#pone.0051426.s009" target="_blank">S9</a>.