Ure 7. NOB1 expression examined by Affymetrix Genechip, and mRNA and protein levels in glioma samples. (A) The expression signal corresponding to NOB1 was significantly higher in high-grade glioma samples compared with low-grade glioma (P = 0.01) and normal brain samples (P,0.001), although the difference between low-grade glioma and normal brain was not statistically significant (P = 0.100). Differences between groups were assessed by one-way ANOVA with the LSD method (*P,0.05). (B) Quantitative RT-PCR showed that NOB1 mRNA was upregulated in low grade glioma (LGG) and high grade glioma (HGG) tissue samples (P = 0.017 and P = 0.032, respectively) compared with normal brain tissue samples from 7 volunteers. (C) Glioma patients who lived more than 24 months (23 patients, 41.8 ) showed decreased NOB1 mRNA expression, whereas patients who lived less than 24 months (32 patients, 58.2 ) showed higher NOB1 mRNA expression (P,0.01) regardless of glioma grade. (D) In patients with LGG, those who lived more than 24 months (13 patients, 52 ) showed lower NOB1 mRNA expression, whereas those who lived less than 24 months (12 patients, 48 ) showed higher NOB1 mRNA expression (P = 0.028). (E) In patients with HGG, those who livedMicroRNA-326 as a Tumor Suppressor in Gliomamore than 24 months (10 patients, 33 ) showed lower NOB1 mRNA expression, whereas those who lived less than 24 months (20 patients, 67 ) showed higher NOB1 mRNA expression (P,0.01). The relative expression of NOB1 mRNA in 1315463 each group was expressed as mean 6 SE, and the differences between groups were determined using the Mann-Whitney U test (*P,0.05. **P,0.01). doi:10.1371/journal.pone.0068469.gColony Formation AssayBriefly, 0.5 mL under layers consisting of 0.8 agar medium was prepared in 6-well plates. A172 or U373 cells with different treatment separately were trypsinized, centrifuged, resuspended in 0.4 agar medium (equal volumes of 0.8 Noble agar and culture medium), and plated onto the top agar at 200 cells per well. The cells were kept for growth for 14 days at 37uC. Colonies were visualized using cell staining Giemsa solution (Chemicon) and counted under the microscope.UK) software. Results were normalized to net integrated pixel density of kit-supplied internal positive controls.Immunohistochemical Staining and EvaluationImmunohistochemical staining for NOB1 protein was performed on the validating set of glioma patients. Briefly, paraffin embedded slides were treated by hydrogen 61177-45-5 peroxide (H2O2) to block endogenous peroxidase activity, and then washed with ddH2O and PBS. Diluted Rabbit polyclonal to NOB1 (Abcam, Cat. #ab87151) was then added for protein binding at room temperature for 60 min. The slides were washed with PBS, incubated with biotinylated secondary antibody (Abcam), and treated with Immunopure Metal enhanced DAB substrate kit (Pierce, Rockford IL) according to the manufacturer’s instructions. Staining was categorized into four grades according to immunohistochemical scores. Briefly, for each slide, 10 randomly selected fields of view under a light microscope were examined for the average intensity of positive cells and then the intensity scores were ��-Sitosterol ��-D-glucoside biological activity assigned to each sample as follows: none (2), none/weak (+2), weak (+); intermediate (++), and strong (+++).Nude Mouse XenograftsNude mouse xenografts were performed as previously reported [16]. Specific pathogen-free six-week-old female BALB/C-nu/nu mice were purchased from the Cancer Research Center of Shanghai.Ure 7. NOB1 expression examined by Affymetrix Genechip, and mRNA and protein levels in glioma samples. (A) The expression signal corresponding to NOB1 was significantly higher in high-grade glioma samples compared with low-grade glioma (P = 0.01) and normal brain samples (P,0.001), although the difference between low-grade glioma and normal brain was not statistically significant (P = 0.100). Differences between groups were assessed by one-way ANOVA with the LSD method (*P,0.05). (B) Quantitative RT-PCR showed that NOB1 mRNA was upregulated in low grade glioma (LGG) and high grade glioma (HGG) tissue samples (P = 0.017 and P = 0.032, respectively) compared with normal brain tissue samples from 7 volunteers. (C) Glioma patients who lived more than 24 months (23 patients, 41.8 ) showed decreased NOB1 mRNA expression, whereas patients who lived less than 24 months (32 patients, 58.2 ) showed higher NOB1 mRNA expression (P,0.01) regardless of glioma grade. (D) In patients with LGG, those who lived more than 24 months (13 patients, 52 ) showed lower NOB1 mRNA expression, whereas those who lived less than 24 months (12 patients, 48 ) showed higher NOB1 mRNA expression (P = 0.028). (E) In patients with HGG, those who livedMicroRNA-326 as a Tumor Suppressor in Gliomamore than 24 months (10 patients, 33 ) showed lower NOB1 mRNA expression, whereas those who lived less than 24 months (20 patients, 67 ) showed higher NOB1 mRNA expression (P,0.01). The relative expression of NOB1 mRNA in 1315463 each group was expressed as mean 6 SE, and the differences between groups were determined using the Mann-Whitney U test (*P,0.05. **P,0.01). doi:10.1371/journal.pone.0068469.gColony Formation AssayBriefly, 0.5 mL under layers consisting of 0.8 agar medium was prepared in 6-well plates. A172 or U373 cells with different treatment separately were trypsinized, centrifuged, resuspended in 0.4 agar medium (equal volumes of 0.8 Noble agar and culture medium), and plated onto the top agar at 200 cells per well. The cells were kept for growth for 14 days at 37uC. Colonies were visualized using cell staining Giemsa solution (Chemicon) and counted under the microscope.UK) software. Results were normalized to net integrated pixel density of kit-supplied internal positive controls.Immunohistochemical Staining and EvaluationImmunohistochemical staining for NOB1 protein was performed on the validating set of glioma patients. Briefly, paraffin embedded slides were treated by hydrogen peroxide (H2O2) to block endogenous peroxidase activity, and then washed with ddH2O and PBS. Diluted Rabbit polyclonal to NOB1 (Abcam, Cat. #ab87151) was then added for protein binding at room temperature for 60 min. The slides were washed with PBS, incubated with biotinylated secondary antibody (Abcam), and treated with Immunopure Metal enhanced DAB substrate kit (Pierce, Rockford IL) according to the manufacturer’s instructions. Staining was categorized into four grades according to immunohistochemical scores. Briefly, for each slide, 10 randomly selected fields of view under a light microscope were examined for the average intensity of positive cells and then the intensity scores were assigned to each sample as follows: none (2), none/weak (+2), weak (+); intermediate (++), and strong (+++).Nude Mouse XenograftsNude mouse xenografts were performed as previously reported [16]. Specific pathogen-free six-week-old female BALB/C-nu/nu mice were purchased from the Cancer Research Center of Shanghai.
