Rized using western blot analysis. After blocking with 5 nonfat milk in

Rized using western blot analysis. After blocking with 5 nonfat milk in PBST for 1 h at room temperature. The rabbit polyclonal antibody against NOB1 (1:10000, Abcam, Cat. #ab87151) or GAPDH (1:5000, Santa Cruz Biotechnology) was incubated with blot overnight at 4uC. Secondary antibody conjugated with horseradish peroxidase (1:10,000; Santa Cruz Biotechnology) was appliedMicroRNA-326 as a Tumor Suppressor in GliomaFigure 3. Cell viability and proliferation were examined in human glioma cells treated with miR-326 precursor. A172 (A) and U373 (B) cells were transfected with miR-326 precursor, control precursor, NOB1 shRNA or nothing for 72 h as described in the methods section before measurement of the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to a colored formazan product. A statistically significant delay of cell proliferation was observed after day 3. A172 cells (C) and U373 cells (D) were transfected with miR-326 precursor, control precursor, NOB1 shRNA or nothing for 72 h as described in the methods section, and the BrdU incorporation assay was performed. BrdU incorporation was decreased in the miR-326 group and NOB1-shRNA group compared to the controls at 72 h. Data represent the mean 6 SD of three independent experiments. Significant differences between transfected cells and mock-infected cells were determined using the two-tailed Student’s t-test (*P,0.05, **P,0.01). doi:10.1371/journal.pone.0068469.gfor 1 h at room 18204824 temperature. Blots were developed using ECL (PE LifeScience).Cell Cycle Analysis by Flow CytometryDifferent cell cycle 23148522 phases (G1, S or G2/M phase) are characterized by different DNA contents. Fluorescence dye propidium iodide (PI) (Sigma, USA) binds to DNA strongly at a ratio of 1:1; hence the DNA contents of cell cycle phases are reflected by Ls as evident from Annexin-V immunofluorescence staining studies. The potential of varying PI fluorescent intensities. Cells were serum starved for 24 h to synchronize the cells, and then the culture medium was replaced with complete medium R cells. Transfected ES cells underwent double-selection with the neomycin analogue containing growth factor. After 48 h of incubation, cells were harvested with trypsinEDTA, washed with chilled PBS twice and fixed with 70 ethanol at 220uC for 2 h. The fixed cells were pelleted, re-suspended in PI/RNase/PBS (100 mg/mL PI and 10 mg/mL RNase A) for atleast 30 min at 37uC in dark, then filtered through a nylon mesh of 400 screen meshes. 16106 cells were analyzed by a FACs caliber II sorter and Cell Quest FACS system (BD Biosciences, USA). This experiment was repeated three times and the results were averaged. No less than 10,000 cells were analyzed in each sample. The percentage of cells in G0/G1, S and G2/M phases was determined by (fluorescence-activated cell sorting) FACS Calibur flow cytometer (BD Biosciences, USA).Cell Proliferation AssayBrdU and MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay were used for cell proliferation measurement. After different transfection for 72 hr, A172 or U373 cells were given a 2-h pulse of BrdU (Sigma) at 4 mg/mL. Visualization of new DNA synthesis was revealed by indirectMicroRNA-326 as a Tumor Suppressor in GliomaFigure 4. Cell cycle distribution and apoptosis of human glioma cells with decreased NOB1 in vitro. Percentage of cells in the G1, G2/M and S phases in A172 (A) and U373 (B) cells with different treatments as described in the methods section. Distribution patterns of normal and apoptotic cells in the four groups were determined by Annexin-PI staining(C). miR-326 expression or NOB1 silencing cau.Rized using western blot analysis. After blocking with 5 nonfat milk in PBST for 1 h at room temperature. The rabbit polyclonal antibody against NOB1 (1:10000, Abcam, Cat. #ab87151) or GAPDH (1:5000, Santa Cruz Biotechnology) was incubated with blot overnight at 4uC. Secondary antibody conjugated with horseradish peroxidase (1:10,000; Santa Cruz Biotechnology) was appliedMicroRNA-326 as a Tumor Suppressor in GliomaFigure 3. Cell viability and proliferation were examined in human glioma cells treated with miR-326 precursor. A172 (A) and U373 (B) cells were transfected with miR-326 precursor, control precursor, NOB1 shRNA or nothing for 72 h as described in the methods section before measurement of the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to a colored formazan product. A statistically significant delay of cell proliferation was observed after day 3. A172 cells (C) and U373 cells (D) were transfected with miR-326 precursor, control precursor, NOB1 shRNA or nothing for 72 h as described in the methods section, and the BrdU incorporation assay was performed. BrdU incorporation was decreased in the miR-326 group and NOB1-shRNA group compared to the controls at 72 h. Data represent the mean 6 SD of three independent experiments. Significant differences between transfected cells and mock-infected cells were determined using the two-tailed Student’s t-test (*P,0.05, **P,0.01). doi:10.1371/journal.pone.0068469.gfor 1 h at room 18204824 temperature. Blots were developed using ECL (PE LifeScience).Cell Cycle Analysis by Flow CytometryDifferent cell cycle 23148522 phases (G1, S or G2/M phase) are characterized by different DNA contents. Fluorescence dye propidium iodide (PI) (Sigma, USA) binds to DNA strongly at a ratio of 1:1; hence the DNA contents of cell cycle phases are reflected by varying PI fluorescent intensities. Cells were serum starved for 24 h to synchronize the cells, and then the culture medium was replaced with complete medium containing growth factor. After 48 h of incubation, cells were harvested with trypsinEDTA, washed with chilled PBS twice and fixed with 70 ethanol at 220uC for 2 h. The fixed cells were pelleted, re-suspended in PI/RNase/PBS (100 mg/mL PI and 10 mg/mL RNase A) for atleast 30 min at 37uC in dark, then filtered through a nylon mesh of 400 screen meshes. 16106 cells were analyzed by a FACs caliber II sorter and Cell Quest FACS system (BD Biosciences, USA). This experiment was repeated three times and the results were averaged. No less than 10,000 cells were analyzed in each sample. The percentage of cells in G0/G1, S and G2/M phases was determined by (fluorescence-activated cell sorting) FACS Calibur flow cytometer (BD Biosciences, USA).Cell Proliferation AssayBrdU and MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay were used for cell proliferation measurement. After different transfection for 72 hr, A172 or U373 cells were given a 2-h pulse of BrdU (Sigma) at 4 mg/mL. Visualization of new DNA synthesis was revealed by indirectMicroRNA-326 as a Tumor Suppressor in GliomaFigure 4. Cell cycle distribution and apoptosis of human glioma cells with decreased NOB1 in vitro. Percentage of cells in the G1, G2/M and S phases in A172 (A) and U373 (B) cells with different treatments as described in the methods section. Distribution patterns of normal and apoptotic cells in the four groups were determined by Annexin-PI staining(C). miR-326 expression or NOB1 silencing cau.

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