Figure 3.

VHL inhibitor stabilizes VHL proteins at a post-translation level. VHL mRNA expressions in (A) HeLa cells treated with 0.5% DMSO, hypoxia (1% O₂), and 250 μM IOX2, 250 μM VH032, or 100 μM VH298 for 16 h or in (B) HFF cells treated with 0.5% DMSO or 100 μM VH298 for 24 h. mRNA was collected, reverse transcribed, and analyzed by quantitative RT-PCR. The shown levels of the indicated mRNAs were normalized to those of β-actin. Graphs depict the mean + SEM of three independent biological replicates. Two-tailed Student's t test was performed to calculate p values, and levels of significance are denoted as follows: ns is p > 0.05. C, HeLa cells treated with 20 μM MG132 for 3 h. 786-O cell lysate was loaded as negative control for VHL and HIF-1α. D, half-life of VHL proteins of HeLa cells treated with DMSO negative control or VH298 VHL inhibitor was measured by treating cells with cycloheximide (CHX) for indicated times. E, protein levels of VHL30 and VH19 were quantified from multiple blots of different exposure time by ImageJ and plotted. Protein levels were analyzed by immunoblotting using antibodies against HIF-1α, VHL, and β-actin, which acted as a loading control. The blots shown are representative of three independent experiments. DMSO, dimethyl sulfoxide; HFF, human foreskin fibroblast; HIF-1α, hypoxia-inducible factor-1α; VHL, von Hippel–Lindau.