Journal: PLoS ONE

Article Title: The Transcription Factor NFAT5 Is Required for Cyclin Expression and Cell Cycle Progression in Cells Exposed to Hypertonic Stress

doi: 10.1371/journal.pone.0005245

Figure Lengend Snippet: A) Dot plots representing γH2AX (H2AX phosphorylated in Ser 139) and DNA content in live NFAT5 +/+ and NFAT5 −/− lymphocytes after 24 hours in isotonic or hypertonic conditions. Bars on the right represent the percentage of γH2AX + cells after 24 hours in isotonic or hypertonic medium (values are the mean±SEM of seven independent experiments; * = p<0.05). B) T cells grown in isotonic or hypertonic conditions during 24 hours were stained with the DNA dye Hoechst 33342 and annexin-V-Fluos, and analyzed by flow cytometry. The experiment shown is representative of three independently performed. C) Single-cell alkaline gel electrophoresis assay (comet assay) done on the population of live cells isolated from cultures of NFAT5 −/− and wild-type cells after 6 or 24 hours in isotonic or hypertonic conditions. Etoposide-treated wild-type cells are included as a positive control. The experiment shown is representative of three independently performed. D) Cell cycle distribution of viable, γH2AX-negative cells after 24 hours in hypertonic medium (representative of seven independent experiments).

Article Snippet: Rabbit polyclonal anti-phospho-p53 (Ser15) (Cat. 9284), mouse monoclonal anti-p53 (Cat. 2524) and mouse monoclonal anti-cyclin D3 (Cat. 2936) were from Cell Signaling Technology (Danvers, MA, USA); mouse monoclonal anti-phospho-histone H2AX (Ser139, γH2AX) (Cat. 05-636) was purchased from Upstate Technologies (Lake Placid, NY, USA); mouse monoclonal anti-BrdU antibody (Cat. 555627) was purchased from BD Pharmingen (San Diego, CA, USA).

Techniques: Staining, Flow Cytometry, Nucleic Acid Electrophoresis, Single Cell Gel Electrophoresis, Isolation, Positive Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Downregulation of lncRNA X Inactive Specific Transcript (XIST) Suppresses Cell Proliferation and Enhances Radiosensitivity by Upregulating mir-29c in Nasopharyngeal Carcinoma Cells

doi: 10.12659/MSM.905370

Figure Lengend Snippet: Effect of XIST knockdown on cell proliferation and radiosensitivity of NPC cells. ( A ) Transfection efficiency of si-XIST-1 and si-XIST-2 in CNE1 and CNE2 cells was detected by qRT-PCR. ( B ) CCK-8 assay was performed to determine cell proliferation at 24 h, 48 h, and 72 h in si-XIST- or si-con-transfected CNE1 and CNE2 cells. ( C ) Colony formation assay was used to measure colony survival rate 2 weeks after CNE1 and CNE2 cells transfected with si-XIST or si-con were exposed to the indicated single doses of irradiation (0, 2, 4, 6, or 8 Gy). ( D ) CNE1 and CNE2 cells transfected with si-XIST or si-con were irradiated with 4-Gy X-rays and then subjected to immunofluorescent staining for γ-H2AX expression. ( E ) Western blot analysis of γ-H2AX expression level in CNE1 and CNE2 cells transfected with si-XIST or si-con under 4 Gy irradiation. * P <0.05.

Article Snippet: After being blocked with 5% nonfat milk in TBST for 1 h at room temperature, the membranes were incubated with primary antibodies, including mouse monoclonal anti-γ-H2AX (No. 2577; 1: 1000 dilution, Cell Signaling Technology, Beverly, MA) and mouse monoclonal anti-GAPDH (No. 97166; 1: 2000 dilution, Cell Signaling Technology) at 4°C overnight, and then by probed with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (sc-2379; 1: 1000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h. Reactive protein expression was visualized and detected using a chemiluminescence kit (Millipore).

Techniques: Transfection, Quantitative RT-PCR, CCK-8 Assay, Colony Assay, Irradiation, Staining, Expressing, Western Blot

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Downregulation of lncRNA X Inactive Specific Transcript (XIST) Suppresses Cell Proliferation and Enhances Radiosensitivity by Upregulating mir-29c in Nasopharyngeal Carcinoma Cells

doi: 10.12659/MSM.905370

Figure Lengend Snippet: Effect of miR-29c overexpression on cell proliferation and radiosensitivity of NPC cells. ( A ) CCK-8 assay was carried out to evaluate cell proliferation at 24 h, 48 h, and 72 h in CNE1 and CNE2 cells transfected with miR-29c or miR-con. ( B ) The clonogenic survival curves were compared in CNE1 and CNE2 cells transfected with miR-29c or miR-con with the indicated single doses of irradiation (0, 2, 4, 6, or 8 Gy) treatment. ( C ) Immunofluorescent staining for γ-H2AX expression in CNE1 and CNE2 cells transfected with miR-29c or miR-con under 4-Gy irradiation. ( D ) Western blot analysis of γ-H2AX level in CNE1 and CNE2 cells transfected with miR-29c or miR-con with 4-Gy irradiation. * P <0.05.

Article Snippet: After being blocked with 5% nonfat milk in TBST for 1 h at room temperature, the membranes were incubated with primary antibodies, including mouse monoclonal anti-γ-H2AX (No. 2577; 1: 1000 dilution, Cell Signaling Technology, Beverly, MA) and mouse monoclonal anti-GAPDH (No. 97166; 1: 2000 dilution, Cell Signaling Technology) at 4°C overnight, and then by probed with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (sc-2379; 1: 1000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h. Reactive protein expression was visualized and detected using a chemiluminescence kit (Millipore).

Techniques: Over Expression, CCK-8 Assay, Transfection, Irradiation, Staining, Expressing, Western Blot

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Selective inhibition of carbonic anhydrase-IX by sulphonamide derivatives induces pH and reactive oxygen species-mediated apoptosis in cervical cancer HeLa cells

doi: 10.1080/14756366.2018.1481403

Figure Lengend Snippet: Representative DNA damage microscopy images from HeLa cell treated with sulphonamide 1 . A fragment of each cell was fixed and processed for γ-H2AX immunofluorescent staining (IFA). γ-H2AX staining is green; nuclei are stained with DAPI blue.

Article Snippet: They were washed with PBS, and 1% BSA-containing PBS was then added, and incubation with primary mouse monoclonal anti-γ-H2AX antibody (Cell Signaling Technology, Danvers, MA) was applied at 37 °C for 1 h. The cells were washed with PBS for 5 min three times.

Techniques: Microscopy, Staining