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. 2015 Apr;14(4):1064–1078. doi: 10.1074/mcp.M114.043885

Fig. 4.

Fig. 4.

FBXO3 is required for mDia2 to stimulate p53-mediated gene transcription in a conformation- and actin-nucleation-independent manner. A, Wild-type mDia2 increases the transcriptional activity of p53 more efficiently than its MA mutant. 293T cells were cotransfected with the reporter plasmid and the empty vector (−), Flag-tagged mDia2 WT (WT) or mDia2 MA (MA). Luciferase activity (arbitrary units, a.u.) was measured and plotted as described in the Experimental Procedures. Data represent mean ± S.D. (n = 12; One-way ANOVA). mDia2 expression was confirmed using anti-Flag antibodies and actin served as a loading control. B, Actin-nucleation-deficient mDia2 activates p53 as efficiently as wild-type mDia2. Cells were transfected and luciferase activity measured and plotted as in A (n = 9; One-way ANOVA). I704 was replaced by A in both wild-type and MA mDia2 to generate IA and IAMA, respectively. C, Generation of control and FBXO3 knockdown cells. 293T cells were infected with either control (shCtr) or FBXO3-targeting (shFBXO3) viruses. Total RNA and lysates were obtained as indicated in the Experimental Procedures. Bar graph: RT-qPCR shows the relative FBXO3 levels (Relative FBXO3 mRNA, arbitrary units (a. u.)) (n = 3; Repeated t Test). Blots: Total cell lysates (30 μg) were immunoblotted as indicated with actin providing a loading control. FBXO3 is reduced by 51.2 ± 3% (shFBXO3 #1) after normalization against actin. One of three experiments that were performed with similar results is shown. D, mDia2-mediated activation of p53 requires FBXO3. Control (shCtr) and FBXO3 (shFBXO3) knockdown cells were cotransfected with the reporter plasmid along with the empty vector (−) or Flag-tagged wild-type mDia2 (+) and luciferase activity measured and plotted as in A (n = 9; One-way ANOVA). E, mDia2 and FBXO3 jointly enhance p53 expression and activity. U2OS cells were cotransfected with the reporter plasmid, Flag-tagged wild-type mDia2 (+) or the empty vector (−), and either HA-tagged FBXO3 (+) or its corresponding empty vector (−). Luciferase activity was measured and plotted as in A (n = 15; One-way ANOVA). p53, mDia2 and FBXO3 expression was confirmed using anti-p53, anti-Flag and anti-HA antibodies, respectively.