FIGURE 3.

VEGFR-2 decrease is due to transcriptional down-regulation. A, VEGFR-2 down-regulation was not due to lysosomal degradation. siRNA-transfected HUVECs were treated with pepstatin at 10 μm for 24 h. Forty-eight hours after transfection cells were lysed and analyzed by Western blot. B, the VEGFR-2 mRNA level was decreased. Forty-eight hours after siRNA transfection, HUVECs were harvested for mRNA extraction. cDNA was obtained by reverse transcription, and real-time PCR was performed. C, mRNA stability was unaffected. Forty-eight hours after siRNA transfection, cells were treated with 7.5 μg/ml actinomycin D at different time points, the mRNA was harvested, and the procedure was repeated. The data represented here are the average of three independent results. D, a non-secretive form of VEGF-A up-regulates VEGFR-2 mRNA level. HUVECs were infected by retroviral overexpression system, and 48 h later mRNAs were extracted. Real-time PCR were realized with reverse-transcribed cDNA. E and F, HUVECs were first transfected by siRNA and 6 h later a retrovirus overexpressing non-secretive form of VEGF-A was added. 72 h after siRNA treatment, mRNAs were extracted for real-time PCR or cells were lysed for Western blot. All bars represent the means ± S.D. of three experiments. *, p < 0.05.