TIP-1 and Radioresistance of Malignant Gliomas V and propidium iodide (PI) (BD pharmingen, San Diego, CA) and analyzed with flow cytometry [25]. antibody (FL393) for SDS-PAGE and immunoblot analysis with a mouse antibody against ubiquitine. c-H2AX foci staining Cells cultured on sterile glass slides were exposed to a mock treatment or 5 Gy X-ray. After the specific time points after the treatment, the cells were fixed and stained with mouse anti– phospho-c-H2AX antibody (Abcam, 1:500), followed by anti- mouse Alexa Fluor 488–conjugated antibody (Invitrogen, 1:2000), or anti-mouse Alexa Fluor 594–conjugated antibody (Invitrogen, 1:2000). DAPI (49,6-diamidino-2-phenylindole) staining was used to visualize the cell nucleus. The images were acquired by using a fluorescent microscope (6400; Carl Zeiss). Quantification of the c- H2AX foci was conducted as previously described [28], as cells containing more than 5 foci were counted as positive. At least 100 cells from each of the triplicate experiments were counted. Comet assay Cells were exposed to 5 Gy X-ray or a mock treatment. After 10 minutes, 4 and 24 hours post irradiation, the cells were collected and combined with low melting agarose before being applied onto CometSlides for electrophoresis according to the manufacturer’s instructions (Cell biolabs, San Diego, CA). Cells were then visualized using fluorescent microscopy (Carl Zeiss). At least 75 comet images were obtained for each time point and analyzed using Comet Score software (Comet Assay IV). Percentages of cells with comet tail as well as comet Tail Moment were calculated upon more than 100 cells from each of three independent experiments [29]. Yeast two-hybrid screening TIP-1 interacting proteins were identified through a yeast two- hybrid screening of human fetal brain cDNA libraries. Screening, hits validation, and identification were performed at the Duke University yeast two-hybrid screening facility (Durham, NC). Pull-down and co-immunoprecipitation Cells were harvested and resuspended in lysis buffer (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P- 40 supplemented with protease inhibitor cocktails and phospha- tase inhibitor cocktail 1 and 2). After the cell debris were removed, 50 mg of the purified protein (GST, GST-tagged wild type or mutant TIP-1) were incubated with the cell lysates for 2 hours at 4uC, followed by incubation with 75 ml GST-binding resins (Novagen, Gibbstown, NJ) for another 2 hours, respectively. After two washes with the lysis buffer, the resin-associated proteins were eluted with 10 mM glycine (pH 2.0). The samples were then subjected to SDS-PAGE and immunoblot analyses. In the immunoprecipitation assays, 500 mg of cell lysate and 2 mg of antibody were incubated with 100 ml of Protein A plus G agrose slurry (Santa Cruz Biotech) at 4uC for 2 hours, the co- immunoprecipitated proteins were analyzed with specific antibod- ies after SDS-PAGE fractionation. p53 ubiquitination assay p53 ubuquitination was detected as previously described [30]. Briefly, at 8 hours post irradiation (5 Gy or mock treated), the cells were incubated with 25 mM proteasome inhibitor MG132 (Sigma) for 4 more hours before the cells were suspended in a lysis buffer (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40 supplemented with protease inhibitor cocktails). p53 was immunoprecipitaed from the cell lysate with a rabbit anti-p53 Tumor growth studies One million of the disaggregated tumor cells within 50 mL of phosphate buffered saline (PBS) were subcutaneously inoculated in the both hind limbs of FoxN1-null nude mice (Harlan Laborato- ries, Prattville, AL). Tumor size was measured with caliper in every other days and calculated as described [31]. When tumor reached 0.3 cm in diameter, X-ray radiation was delivered through a Pantak Therapax DXT 300 X- ray unit (Pantak, East Haven, CT) at 3 Gy for five consecutive days. Only the tumors in the right hind limbs were irradiated while the tumors in the left hind limbs of the same animals were used as mock radiation treatment control. Animals were sacrificed when tumor size was bigger than 1 cm in diameter or tumor growth affected the mouse behaviors or motility. All the animal studies were conducted as approved by the Institutional Animal Care and Use Committee (IACUC) at Vanderbilt University. Statistical analyses Microarray data for human glioblastoma multiformes were from ‘‘The Cancer Genome Atalas’’ project [32]. Patients with elevated TIP-1 expression levels (log2 tumor/normal ratio .1.5) are defined as ‘‘TIP-1 high’’ group, those with the ratio less than 0.5 are defined as ‘‘TIP-1 low’’ group. Survival curves were plotted with the Kaplan-Meier method and compared with a log-rank test. The Student’s t test was used to determine the statistical significance unless otherwise stated. All quantitative data was presented as mean +/2 standard deviation. Results Elevated TIP-1 expression levels correlate with the poor prognosis of human malignant gliomas after radiotherapy To determine the prognostic value of the TIP-1 expression in glioma patients after radiotherapy, we analyzed the microarray datasets from ‘‘The Cancer Genome Atalas’’ project [32]. All these brain tumors are glioblastoma multiformes (GBM, grade IV according WHO classification) and express wild-type TIP-1. Patients with elevated TIP-1 expression levels (log2 tumor/normal ratio .1.5) are defined as ‘‘TIP-1 high’’ group, those with the log2 tumor/normal ratio less than 0.5 are defined as ‘‘TIP-1 low’’ group. Kaplan-Meier survival curves were developed for those patients (Fig. 1). Statistical analysis using Log Rank Test indicated that TIP-1 expression levels correlate to the survival probability of GBM patients after radiotherapy (p,0.05), the patients with elevated TIP-1 expression levels experienced a poor prognosis after radiotherapy. TIP-1 knockdown sensitizes glioma cells to X-ray radiation The putative roles of TIP-1 expression in the cellular response of glioma cells to ionizing radiation were studied with clonogenic assays. Stable cell lines with ectopic TIP-1 expression or shRNA downregulation were established, and the TIP-1 protein expres- sion in those cells was verified by western blot analyses of the cell lysates with specific antibodies (Figs. 2A). Two independent shRNA sequences for TIP-1 knockdown were used to prevent bias from off-target effects. In the clonogenic assays, the ectopic expression or depletion of TIP-1 did not affect the cell plating efficiency (data not shown). Compared to the vector control or PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e45402