Fig. 2

Gossypol induces mouse double minute 2 (MDM2) self-ubiquitination and protein-degradation. a MCF-7 and MDA-MB-468 cells were treated with 10 μM gossypol for different time periods. The mRNA levels of MDM2 in tumor cells were determined by quantitative RT-PCR. Data represent means ± SEM of three independent experiments normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). b MDA-MB-468 cells were transfected with MDM2 promoter luciferase plasmids (MDM2 p1-Luc or p2-Luc). Transfected cells were treated with increasing concentrations of gossypol (2.5, 5.0, 7.5, and 10.0 μM) for 4 h and then analyzed by luciferase activity assay. c MDA-MB-468 cells were treated with or without 10 μM gossypol for 4 h, followed by addition of 5 mg/ml actinomycin D. At different time points indicated after addition of the mRNA synthesis inhibitor actinomycin D, the cells were harvested and the amount of MDM2 mRNA in the cells was detected by quantitative RT-PCR. d MDA-MB-468 cells were treated with or without 10 μM gossypol for 8 h, and then their cytoplasmic lysates were fractionated on a sucrose gradient. RNA extracted from each of the fractions was subjected to quantitative RT-PCR for quantitative analysis of the distribution of MDM2 mRNA. Data represent percentage of the total amount of corresponding mRNA for each fraction. e MDA-MB-468 cells were treated with or without 10 μM gossypol for 4 h, followed by addition of a protein synthesis inhibitor cycloheximide (CHX, 50 μg/ml). At different time points after CHX, cell lysates were prepared and analyzed by western blot assay. f Ubiquitination assay in MDA-MB-468 cells for testing the effects of increasing concentrations of gossypol (2.5, 5.0, and 10.0 μM) on ubiquitination of MDM2 in the presence or absence of VEGF 3′UTR. g MDA-MB-468 cells were treated with a proteasome inhibitor MG132 (30 μM) for 4 h and subsequently with 10 μM gossypol for the indicated times. MDM2 ubiquitination was analyzed by immunoprecipitation-western blot assay. h, i Immunoprecipitation-western blot assay to detect the effect of gossypol on the ubiquitination of mut-MDM2 C464A without E3 ligase activity. VEGF vascular endothelial growth factor, wt wild-type