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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 6. — Blocking Rac1 function largely abolishes mutant p53 GOF in promoting anchorage-independent growth of tumor cells and growth of xenograft tumors. (A–C) Blocking Rac1 activity greatly abolished mutant p53 (R175H and R273H) GOF in promoting anchorage-independent growth of H1299 cells in soft agar. (A) H1299 cells expressing mutant p53 (R175H and R273H) and control H1299-Con cells were transfected with shRNA vectors against Rac1. (Left panels) Representative images. Bar, 200 µm. (Right panels) Average colony numbers in soft agar. (B) Cells were transfected with vectors expressing Rac1 DN. (C) Cells were treated with 10 µM NSC23766 or PBS. (D) Expression of Rac1-R but not Rac1^ΔSUMO-R in H1299 cells with knockdown of endogenous Rac1 largely restored mutant p53 GOF in promoting anchorage-independent cell growth in soft agar. (E,F) Blocking Rac1 activity by shRNA vectors against Rac1 (E) or 10 µM NSC23766 treatment (F) greatly inhibited anchorage-independent cell growth in soft agar for SK-BR-3 (left panels) and MDA-MB468 (right panels) cells but had a less pronounced effect on SK-BR-3 and MDA-MB468 cells with knockdown of endogenous mutant p53. For A–F, data are presented as mean ± SD. n = 6. (#) P < 0.05; (*) P < 0.01; (**) P < 0.001, Student's t-test. (G) Rac1 knockdown by shRNA vectors greatly inhibited the growth of H1299 mutant p53 (R175H and R273H) xenograft tumors but had a less pronounced effect on H1299-Con tumors. (Left panels) Representative images of xenograft tumors. Bar, 1 cm. (Right panels) Growth curves of xenograft tumors. (H) Blocking Rac1 activity by expression of Rac1 DN largely abolished mutant p53 (R175H) GOF in promoting the growth of H1299 xenograft tumors. (I) Expression of Rac1-R but not Rac1^ΔSUMO-R in H1299 cells with knockdown of endogenous Rac1 largely restored mutant p53 (R175H and R273H) GOF in promoting xenograft tumor growth. (J) Ki-67 IHC staining of H1299-Con and H1299 mutant p53 (R175H and R273H) xenograft tumors with or without Rac1 knockdown. (Left panels) Representative images. Bar, 20 µm. (K) The levels of Rac1 and p-PAK1/2 in H1299 mutant p53 (R175H) xenograft tumors as examined by Western blot assays. (L) Blocking Rac1 activity by NSC23766 treatment largely abolished mutant p53 (R175H) GOF in promoting the growth of H1299 xenograft tumors. Mice were treated with 2.5 mg/kg NSC23766 intraperitoneally once every 2 d for 2 wk starting from day 6 after cell inoculation. For G–I, and L, tumor volumes are presented as mean ± SD. n = 6 per group. (**) P < 0.001, ANOVA followed by Student's t-test.