Figure 5. NQO1 stabilizes HIF-1α through inhibiting ubiquitination and proteasome-mediated degradation.

(a,b) RKO (a) and MDA-MB-231 (b) cells were transfected with pshCont or pshNQO1 and pCDNA3.1-myc-His₆ or pNQO1-myc-His₆, respectively. The cells were then exposed to 0.5% O₂ for 2 h, and incubated for 1 h in the presence or absence of MG132. Whole-cell extracts were immunoprecipitated with an anti- HIF-1α antibody, and ubiquitinated HIF-1α was detected with an anti-ubiquitin antibody. (c,d) RKO (c) and MDA-MB-231 (d) cells were exposed to 20 or 0.5% O₂. After 2 h, the cells were treated with or without cycloheximide and MG132, incubated for 1 h, and harvested. The collected cells were analysed by immunoblotting for HIF-1α, NQO1 and β-actin. (e,f) RKO cells (e) and MDA-MB-231 cells (f) were exposed to 0.5% O₂. After 2 h, the cells were harvested and immunoprecipitated with anti- HIF-1α or anti-IgG (negative control) antibodies. The precipitates were analysed by immunoblotting with the indicated antibodies. (g) RKO/pshCont and RKO/pshNQO1 cells were treated with or without 1 mM of DMOG for 1 h, and then exposed to 0.5% O₂. After 2 h, the cells were harvested and analysed by immunoblotting with the indicated antibodies. (h) RKO/pshCont and RKO/pshNQO1 cells were transfected with siPHD1, siPHD2, siPHD3 or HIF-1α. After 48 h, the cells were treated with 50 μg MG132 for 6 h, exposed to 0.5% O₂ for another 2 h, and then harvested. Whole-cell lysates were analysed by immunoblotting for HIF-1α, hydroxylated-HIF-1α, PHD1, PHD2, PHD3, NQO1 and β-actin. X-ray films were exposed for 1, 3 or 5 min to detect the signals of total HIF-1α, hydroxylated-HIF-1α.