Article title: Reactive oxygen species production has a critical role in hypoxia-induced Stat3 activation and angiogenesis in human glioblastoma

Journal name: Journal of Neuro-Oncology

Author names: Mi Ok Yu, Kyung-Jae Park, Dong-Hyuk Park, Yong-Gu Chung, Sung-Gil Chi, and Shin-Hyuk Kang

Corresponding Authors:

Shin-Hyuk Kang, M.D., Ph.D.

Department of Neurosurgery, College of Medicine, Korea University #126, 5-ga, Anam-Dong, Seongbuk-Gu, Seoul, 136-705, Korea

Tel: +82-2-920-5729, Fax: +82-2-929-0629, E-mail:

Sung-Gil Chi, Ph.D.

School of Life Sciences and Biotechnology, Korea University, 5-1 Anam-Dong, Seongbuk-Gu, Seoul 136-701, Korea

Tel: +82-2-3290-3443, E-mail:

Materials and Methods

Cell culture and Hypoxia

Human glioblastoma cell lines (U87, U373, A172 and U251) were maintained in plastic flasks as adherent monolayers in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. All cell cultures were maintained under an atmosphere of 5% CO2 in 95% humidity at 37°C. To induce hypoxic condition, the cells were incubated in an anaerobic incubator (ASTEC, Fukuoka, Japan) at 94% N2, 5% CO2 and 1% O2.

Ribonucleic Acid Isolation and RT-PCR

Total RNA was separately extracted from the cell lines using the TRIzol reagent (Invitrogen) and reverse transcribed with the Super Script kit (Invitrogen) according to the manufacturer's protocol. Real-time reverse transcription polymerase chain reaction (RT-PCR) and semi-quantitative RT-PCR were performed in the icycleriQ multicolor real-time PCR detection system (Bio-Rad, Richmond, California) and GeneAmp PCR system (Applied Biosystems), respectively.Each sample was run in triplicate and results were normalized by their corresponding GAPDH content. Primer sequences used in this study were shown in Table 1.

Immunofluorescence analysis

U87 and U373 cells were plated on culture slide and allowed to adhere overnight. After 24 hours, cells were incubated under the hypoxic condition for the indicated time. The cells were then fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked with phosphate buffed saline containing 3% bovine serum albumin. The slides were incubated with phosphorylated Stat3 (Tyr705) antibody (Cell Signaling Technology) diluted 1:50 and Nox4 antibody (abcam) diluted 1:100 in blocking solution overnight at 4°C.Cy3 anti-rabbit IgG (Jackson ImmunoResearch Laboratories) diluted 1:1000 in blocking solution was used as secondary antibody. The cells were photographed and analyzed with confocal microscope (Carl ZeissMicrosImaging).

Immunoblot assay

25㎍of whole-cell lysates were separated on 10% sodium dodecyl sulfate/polyacrylamide gel and transferred onto PVDF membranes. The membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.05% Tween20 at room temperature for 30 minutes and incubated overnight at 4°C with anti-phosphorylated-Stat3 (Tyr705), anti-Stat3 (Cell Signaling Technology), anti-Nox4 (abcam), anti-HIF-1(Novus) and anti-beta-actin (BioVision Research Products)antibodies diluted 1:2000 in 3% bovine serum albumin. The anti-rabbit IgG and anti-mouse IgG (Jackson ImmunoResearch Laboratories) were used as a secondary antibody diluted 1:20000 in 1% nonfat milk. The probed proteins were detected by use of the chemiluminescence system (Thermo scientific) according to manufacturer instructions.

Measurement of ROS

To quantify ROS caused by hypoxia, the cells were incubated under the hypoxic condition and stained with 5uM Mitosox, 30uM 2’, 7’-Dichlorofluorescein diacetate(DCFDA)or 10uM dihydroethidium(DHE) for 30 minutes under normoxic condition, and analyzed by a Becton Dickinson FACS calibur or confocal microscope (Carl ZeissMicroImaging).

Enzyme-Linked Immunosorbent Assay

U87 cells were transfected with either siControlor siNox4-1 and subjected to either normoxic or hypoxic condition. Conditioned media were collected and VEGF concentration was measured using the quantikine human VEGF immunoassay kit (R&D Systems) in accordance with the manufacturer’s instructions.

Tube formation

U87 cells were transfected with siControl or siNox4-1 and incubated for 48 hours and then incubated for 20 hours under hypoxic condition. After that conditioned media were collected and stored frozen at -20°C. 100㎕growth factor-reduced Matrigel (BD Biosciences) was added into each well of a 96 plate. After polymerization for 30 minutes at 37°C, 1.5 X 104 human umbilical vein endothelial cells (HUVEC) were plated on a layer of polymerized matrigel. Conditioned media were added, and cells were incubated at 37°C, 5% CO2 for 18 hours and photographed. Tube formation was quantified the percentage of the cell surface area vs. the total surface area. HUVEC cultured with conditioned media from siControltransfected and normoxia cultured U87 were assigned arbitrary percentage values of 100.

Statistic analysis

Data are presented as the mean ± standard error of measurement (SEM). The student’s unpaired t-test using Graphpad Prism software was used to calculate statistically significant differences across the groups (P＜0.05).

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