Fig. 4.

Effect of MDM2 and its cytoplasmic location on VEGF translation. (A) SK-N-SH cells were cotransfected with 5 μg pGL3-VEGF 3′UTR (wt or mutant [mut]) plasmids and with increasing amounts (5 and 10 μg) (indicated by the height of the black triangle) of the wt MDM2 plasmid (MDM2/wt), MDM2/166A plasmid, or pcDNA 3.1-HuR plasmid. After 24 h, cell extracts were prepared, and their quantitative FL (pGL3-MYCN 3′UTR) was detected using the Dual-Luciferase reporter system. Only the FL activities in the transfection of pGL3-MYCN 3′UTR were set at 1. Bars represent the means plus SD (error bars) of FL in three independent experiments, after being normalized to the RL activity (cotransfected pRL-SV40 vector was internal control). (B) Comparison of VEGF protein production during hypoxia in SK-N-SH cells that were transfected with wt MDM2, MDM2/166A, or control vector. Equal numbers of transfected cells were exposed to hypoxia, and then at the indicated time point, cell supernatants were harvested to detect VEGF levels by ELISA. Values that are significantly different are indicated by asterisks as follows: *, P < 0.01; **, P > 0.05. (Inset) The expression levels of transfected MDM2s were detected by Western blotting. (C) Representative polyribosomal profiles from MDM2/166E and MDM2/166A-transfected MDM2^−/− p53^−/− MEF cells. The traces obtained during fraction collection at 254 nm are shown. (D and E) Relative distribution of VEGF (D) and GAPDH (E) mRNA in MDM2^−/− p53^−/− MEF cells that were transfected with either MDM2/166E or MDM2/166A. Their cytoplasmic lysates were fractionated on a sucrose gradient. RNA extracted from each fraction was subjected to qRT-PCR. Data represent the percentage of the total amount of corresponding mRNA for each fraction.