Article title: Romo1 is associated with ROS production and cellular growth in human gliomas

Journal name: Journal of Neuro-Oncology

Author names: Mi Ok Yu,Na-Hyun Song, Kyung-Jae Park, Dong-Hyuk Park, Se-Hyuk Kim, Yang-SeokChae,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

Materials and Methods

Cell culture and tissue samples

Six human brain cell lines including four GBM cell lines (U87, T98, U373 and LN229), one glioma cell line (HS683), and immortalized human astrocytes (NHA) and two human cervical cancer cell lines (HeLa and SiHa) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. All cell cultures were maintained under an atmosphere of 95% O2 and 5% CO2 in 98% humidity at 37°C.

Tumors were obtained from patients by surgical resection at the Department of Neurosurgery of Korea University Anam Hospital. Tumors were diagnosed as Grade IV according to the WHO classification.Tumor tissues were immediately soaked in RNAlater RNA Stabilization Reagent and stored in the -80℃ freezer until RNA was extracted.

Knockdown by lentiviral vector-mediated shRNAand overexpression by plasmid transfection

shRNA clones for Romo1 knockdown were obtained by screening lentivirusshRNA sets (Mission shRNA, Sigma-Aldrich). Romo1-targeting and control lentiviruses were produced in HEK293T cells with the MISSION Lentiviral Packaging Mix(Sigma). Four shRNA clones were characterized for knockdown efficiency using a real-time RT-PCR. The most efficient clone was used for all further experiments.

FLAG-tagged Romo1 and empty pFLAG-C1 plasmid were kindly provided by Young Do Yoo (Laboratory of Molecular Cell Biology, Korea University, Republic of Korea). Plasmid was transfected by using Lipofectamine 2000 (Invitrogen) and Romo1 expression was confirmed by semiquantitative RT-PCR Immunofluorescence and immunoblot analysis.

RNA and DNA Isolation and PCR

Total RNA was extracted from cell lines and tissues using the TRIzol reagent (Invitrogen) and reverse transcribed with the Super Script kit (Invitrogen) according to the manufacturer's protocol. A real-time RT-PCR was performed using iQ SYBR Green Supermix (Bio-Rad). Each sample was subjected to PCR for Romo1 and GAPDH in triplicate and the PCR results were normalized by their corresponding GAPDH content. Genomic DNA was isolated by using Genomic DNA kit (invitrogen) and PCR was done. Primer sequences used in this study were shown in Table 1.

Western blot

Whole-cell lysates were prepared from cells as described previously [26]. The lysates were separated on 12% sodium dodecyl sulfate/polyacrylamide gel and transferred onto PVDF membrane. The membranes were probed withanti-Romo1 (OriGene) anti-phospho-cdc2, anti-cdc2, anti-caspase 3(Cell Signaling Technolog), anti-FLAG (Sigma) and anti-beta-actin (BioVisionResearch Products) antibodies. The blots were further probed with secondary antibodies and detected using the ECL detection system (Amersham Life Sciences).

Immunocytochemistry

U87 and U373 cells were plated on culture slides and allowed to adhere overnight. After 24 hours, the cells were infected with either control or Romo1 shRNA-1. The cells were 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 anti-Romo1 antibody diluted 1:50 in blocking solution for 1 hour at room temperature. The Alexa 488 anti-mouse IgG (Invitrogen, San Diego, California) was used as secondary antibody diluted 1:200 and the cells were photographed and analyzed with fluorescence microscope (Olympus LX71 microscope).

Immunohistochemistry

Sections of the 19 tissue microarray block were deparaffinized in xylene, hydrated in graded alcohols and rinsed with PBS. For epitope retrieval, the tissue sections were soaked in citrate buffer at pH 6.0 and heated in a microwave oven for 8 min. After cooling the sections for 15 min on the ice, endogenous peroxidase activity was blocked using hydrogen peroxide/methanoland 10% fetal bovine serum for 30 minutes. The sections were then incubated with mouse anti Romo1 antibody (1:20 dilution), and incubated with anti-mouse IgG. For the detection of bound primary antibodies, the EnVision technique (DakoCytomation; Carpinteria, CA, USA) with diaminobenzidinechromogen as a substrate was used, and the cell nucleus was counterstained with haematoxylin. Staining was classified using a three-tiered system (negative, 0/+; moderate positive, ++; and strong positive, +++) according to the percentage of positive cells and staining intensity as described previously [26]. Scoring of staining intensity was conducted in a blinded manner to prevent bias from knowledge of clinical data.

Measurement of ROS

Quantitation of ROS production was done by using DCF-DA and mitoSOX staining. The cells were infected with either control or Romo1 shRNA-1, stained with 30 uMdichlorofluoresceindiacetate (DCF-DA) for 30 min, and analyzed using a Becton Dickinson FACScan, or stained with 5 uMMitoSOX and 30 uM DCF-DA and analyzed by a fluorescence microscope (Olympus LX71 microscope).

Cell viability and soft agar colony formation assay

Infected cells were stained with trypan blue (Invitrogen) and counted using a hemocytometer at 24, 48, 72 and 96 hours. For colony formation assay, infected cells were seeded in 0.35% agar at a density of 5 × 103 cells per well of 6 well plate and cultured for 20 days at 37°C under 5% CO2. Dishes were stained with 0.01% crystal violet and colonies were counted in triplicate wells from 6 fields photographed.

BrdU assay

BrdU incorporation analysis was performed using a FITC BrdU flow kit (BD PharmingenTM). Cells were treated withBrdU and cultured at 37℃for 45min. The cells were fixed and stained with anti-BrdU and 7-amino-actinomycin D (7-AAD). Cell cycle analysis was carried out using Becton Dickinson FACScan and Cell Quest software.

In vivo tumorigenicity study

U87 cells (3 × 106) infected with either control or Romo1 shRNA-1 were injected subcutaneously into the left flank region of 7-week-old BALB/c nude mice. After 40 days, tumor size was measured every 7 days with a calipers and volume was calculated as (length × width2)/2.

Fig. S1