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Blood, Vol. 111, Issue 6, 3131-3136, March 15, 2008
Bortezomib inhibits tumor adaptation to hypoxia by stimulating the FIH-mediated repression of hypoxia-inducible factor-1
Blood Shin et al.
111: 3131
Supplemental materials for: Shin et al
Files in this Data Supplement:
- Figure S1. Effect of bortezomib on HIF-1α protein or EPO mRNA expression (JPG, 52.7 KB)
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(A) Hep3B cells were incubated under hypoxic conditions for 16 hours in the presence of bortezomib (Bort) at >1 nM concentrations. Cells were lysed in a denaturing SDS sample buffer, and HIF-1a and b-tubulin proteins were analyzed by Western blotting. (B) Hep3B cells were incubated under hypoxic conditions for 16 hours in the presence of bortezomib (Bort) at >1 nM concentrations. Total RNAs were extracted, and EPO mRNA was analyzed by semi-quantitative RT-PCR.
- Figure S2. Proteasome acivity (JPG, 34.3 KB)
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Proteasome activity was analyzed using a proteasome substarate, LLVY, conjugated with the fluorophore 7-Amino-4¬methylcoumarin (AMC), purchased from Chemicon Inc. (Temecula, CA). Hep3B cells were treated with various concentrations of bortezomib or 10 µM of MG132 for 16 h. Cells were lysed in a buffer containing 25 mM HEPES, pH 7.5, 0.5 mM EDTA, 0.1% Nonidet P-40 and 0.002% SDS. Seven ml of cell lysate was incubated with 50 mM LLVY-AMC for 90 min at 37°C, and the free AMC released from the proteasome was quantified using a 380/460 nm filter set in a fluorometer (Cytofluor 2350 plate reader, Millipore). Data are the means of two separate experiments.
- Figure S3. Subcellular localization of HIF-1α (JPG, 81.4 KB)
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Hep3B cells were incubated under normoxic or hypoxic conditions for 16 h in the presence of bortezomib (Bort; 1 nM or 100 nM). The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking nonspecific binding with 5% normal goat serum for 1 h, the cells were incubated with anti-HIF-1 antibody (1:100) at 4°C for 16 h, followed by staining with FITC-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories) for 1 h. The nuclei were counterstained with 0.1 µg/ml of DAPI, and the subcellular localizations of HIF-1 were examined using an IX71 fluorescence microscope (Olympus, Hamburg). Images were captured using an UIS 2 Olympus camera running Image pro Plus 5.1 software (Olympus). The images in the 1st column are captured under white light, and those in the 2nd and 3rd columns are fluorescence images through green and blue filters, respectively. The 4th column shows merged images from green and blue fluorescence.
- Figure S4. Subcellular localization of FIH (JPG, 82.2 KB)
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Hep3B cells were incubated under normoxic or hypoxic conditions for 16 h in the presence of bortezomib (Bort; 1 nM or 100 nM). The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking nonspecific binding with 5% normal goat serum for 1 h, the cells were incubated with anti-FIH antibody (1:100) at 4°C for 16 h, followed by staining with FITC-conjugated secondary antibody for 1 h. The nuclei were counterstained with 0.1 µg/ml of DAPI, and the subcellular localizations of FIH were examined using an IX71 fluorescence microscope and an UIS 2 Olympus camera. The images in the 1st column are captured under white light, and those in the 2nd and 3rd columns are fluorescence images through green and blue filters, respectively. The 4th column shows merged images from green and blue fluorescence.
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