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

Figure 5.

Figure 5.

Mutant p53 inhibits SENP1–Rac1 interaction and SENP1-mediated Rac1 de-SUMOylation in human cancer cells. (A) SENP1 decreased Rac1 SUMOylation levels in H1299 cells. H1299 cells were transfected with vectors expressing Flag-SENP1, Myc-Rac1, and HA-SUMO-1, respectively. (B) The central region of the Rac1 protein is required for SENP1–Rac1 interaction in cells. H1299 cells were transfected with vectors expressing Flag-SENP1 and Myc-Rac1 (full length or with the deletion of the central region), respectively, for co-IP assays. (FL) Full length. (C) Mutant p53 inhibited SENP1–Rac1 interaction in H1299 cells. H1299 cells were transfected with increasing amounts of mutant p53 (R175H or R273H) together with constant Flag-SENP1 and Myc-Rac1 expression for co-IP assays. (D) Knockdown of endogenous mutant p53 (R175H and R273H, respectively) in SK-BR-3 and MDA-MB468 cells increased SENP1–Rac1 interaction in cells. SK-BR-3 and MDA-MB468 cells with or without endogenous mutant p53 knockdown were transfected with vectors expressing Flag-SENP1 and Myc-Rac1, respectively, for co-IP assays. (E) Knockdown of endogenous SENP1 largely abolished the promoting effect of mutant p53 on Rac1 SUMOylation in H1299 cells. H1299 cells with or without endogenous SENP1 knockdown by siRNA oligos were transfected with the indicated combination of vectors expressing mutant p53 (R175H), Myc-Rac1, and HA-SUMO-1, respectively. (F) Knockdown of endogenous SENP1 largely abolished the promoting effect of mutant p53 on Rac1 activation in H1299 cells, which can be restored by expression of SENP1 but not SENP1C603S resistant to the SENP1 siRNA targeting endogenous SENP1 at its 3′ untranslated region (UTR). H1299 cells with or without endogenous SENP1 knockdown by siRNA targeting endogenous SENP1 at its 3′ UTR were transfected with the indicated combination of vectors. (G) Knockdown of SENP1 by siRNA in SK-BR-3 and MDA-MB468 cells largely abolished the promoting effect of endogenous mutant p53 (R175H and R273H) on Rac1 activity, which can be largely restored by expression of SENP1 but not SENP1C603S resistant to the SENP1 siRNA. In F and G, data are presented as mean ± SD. n = 3. (**) P < 0.001, Student's t-test.