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Supplementary Materials
Western blot analysis
Total protein extracts, concentration determination and electrophoresis were prepared as
described (Liu et al., 2008). After blocked in 3% nonfat milk, blots were probed with the
primary anti-POX/HIF-1α antibody (1:1000) followed by secondary antibody conjugated
to horseradish peroxidase (1:2000). All blots were developed using the ECL procedure
(Amersham Biosciences). After stripped, blots were reprobed with anti-actin monoclonal
antibody to serve as controls.
Luciferase reporter assay
786-0 and TK10 renal cancer cells were plated in 24-well plates and cotransfected with
400 ng of pMIR-REPORT, pPOX 3’ UTR or pPOX 3’UTR-MUT and 5 ng of pRL-null
vector (Promega) with or without mimic/mutant miR-23b* or negative siRNA, using
Lipofectamine 2000TM. The cotransfected pRL-null vector that provided the constitutive
expression of renilla luciferase was used as an internal control to correct the differences
in both transfection and harvest efficiencies. Luciferase activity was measured with equal
amounts of cell lysates using the Dual-Luciferase Reporter Assay System (Promega) by a
TD-20/20 luminometer (Turner Designs).
Apoptosis assay
Transfected renal cancer cells were harvested and then treated with annexin-V FITC and
propidium iodide (PI) in the Annexin-V FITC Apoptosis Kit (Invitrogen) followed by
flow cytometric analysis according to the manufacturer’s protocol. The apoptotic
percentage was calculated as the percentage of Annexin-V positive cells in the total cell
number.
Proliferation assay
The CellTiter 96 aqueous nonradioactive cell proliferation assay (Promega) was
performed according to the manufacturer’s protocol.
Measurement of intracellular ROS
Renal cancer cells or DLD1 Tet-off POX cells were seeded in a 6-well plate. The cells
were transfected with anti-23b* and/or siRNA for 48 h before analysis of ROS. For the
measurement, media was removed and washed with PBS. Phenol red-free medium,
containing 50 μM 2,7-dichlorohydro fluorescein diacetate (DCF-DA) was added to the
monolayer, and the plates were incubated at 37°C for 45 minutes. The plates were read
on a CytoFluor 4000 (PerSeptive Biosystems) with excitation wavelength of 485 nm and
emission of 530 nm. Cells were then washed and collected and protein content was
determined as described.
POX imunohistochemical (IHC) staining and miR-23b* in situ hybridization (ISH)
The immunostaining for POX for 16 pairs of kidney normal and tumor tissues was
performed in the Pathology/Histotechnology Laboratory, NCI-Frederick. The dilution of
POX antibody was 1:1000. In situ hybridization for miR-23b* for those samples was
carried out as previously published (Thompson et al., 2007). Fluorescein-hapten labeled
oligonucleotide probe for miR-23b* was used with reversed sequence of miR-23b* as
negative control. Pathologists from the above laboratory helped read and grade the
expression of miR-23b* and POX.