Fig. 4.

Quantitative analysis of the inhibitory effect of Nutlin-3 and cmpd 7 for p53/MDM2 interaction. (A) Chemical structure of Nutlin-3 and cmpd 7 (an inactive fragment of Nutlin family). (B) Indicated pair of fusion proteins were expressed in U2OS cells for 24 hours and then treated with Nutlin-3 and cmpd 7 for 30 min prior to cell fixation and subsequent visualization. The mean intensity of green and red fluorescent signals on the p-body was quantitatively measured, and the relative intensities were presented (n of p-body spots = 41, 52, 34 for DMSO, Nutlin-3, and cmpd 7, respectively). (C) Ten hours after the treatment with Nutlin-3 and cmpd 7 on U2OS cells, the amount of endogenous p53 protein was analyzed by Western blotting. The asterisk indicates a nonspecific band. HnRNP A1 was monitored as a loading control. (D) The p53 and hnRNP A1 proteins from Western blotting were quantified, and the amount of protein relative to that in the DMSO-treated samples are presented. The mean ± s.d. was calculated from three independent experiments.