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. 2017 Aug 15;31(16):1641–1654. doi: 10.1101/gad.301564.117

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© 2017 Yue et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 2. — Mutant p53 activates Rac1 in human cancer cells. (A) Mutant p53 increased Rac1 activity in H1299 cells, represented by increased levels of Rac1-GTP. H1299 cells with or without ectopic mutant p53 (R175H, R248W, or R273H) expression were used to perform the GST-p21-binding domain of PAK1 pull-down assays to specifically pull down Rac1-GTP in cells followed by Western blot assays to measure the Rac1-GTP levels. (B) Ectopic mutant p53 expression increased the levels of p-PAK1^Ser144 and p-PAK2^Ser141 (p-PAK1/2) in H1299 cells. (C) Knockdown of endogenous mutant p53 by shRNA vectors decreased the levels of Rac1-GTP and p-PAK1/2 in SK-BR-3, MDA-MB468, LAPC4, and SW480 cells. Two shRNA vectors against p53 were used for all cell lines, and very similar results were observed. For the sake of clarity, results of one shRNA vector are presented for SW480 and LAPC4 cells. (D) Wild-type p53 decreased Rac1 activity, represented by decreased Rac1-GTP levels in human cancer cells. H1299 cells with or without ectopic wild-type p53 expression, human colorectal cancer HCT116 cells with or without wild-type p53, and MCF7 cells with or without wild-type p53 knockdown were used. For A, C, and D, the top panels show representative Western blot images for Rac1 activity analysis, and the bottom panels show quantitation of relative Rac1-GTP/total Rac1/Actin levels. Data are presented as mean ± SD. n = 3. (*) P < 0.01; (**) P < 0.001, Student's t-test.