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: Gynecologic oncology

Article Title: Panobinostat sensitizes cyclin E high, homologous recombination-proficient ovarian cancer to olaparib

doi: 10.1016/j.ygyno.2016.07.088

Figure Lengend Snippet: A) The percentage of OVCAR-3 and SKOV-3 cells showing co-expression of RAD51 and pH2AX (positive was >5 foci). Cells were pre-treated with 0.5µM cisplatin (6h), then effects of the panobinostat pre-treatment (25nM; 24h)/panobinostat (25nM; 24h) and olaparib (10µM) co-treatment combination regimen (24h) determined by IF. B) IF analysis of GFP expression in cells co-transfected with pDRGFP and I-Sce1 plasmids (both 1µg), then treated with panobinostat and/or olaparib as above. At least 100 cells were counted in 3 independent fields. Values in A) and B) are mean+SD for 3 independent experiments. * p<0.01 compared to vehicle; ap<0.01 relative to olaparib alone, bp<0.01 relative to panobinostat alone, Student’s t test. C) Representative images of GFP-positive SKOV-3 cells (green) and DAPI-stained nuclei (blue). Scale bars are 20µm.

Article Snippet: Immunofluorescence Following drug treatment and/or transient transfection with the HR reporter plasmid pDRGFP and endonuclease encoding pCBASce1 (I-Sce1) (both gifts from Maria Jasin; Addgene plasmids #26475 and #26477, respectively) [ 27 , 28 ] plasmids using Lipofectamine 2000 according to manufacturer’s instructions (Invitrogen Corp., Carlsbad, CA), ovarian cancer cells were fixed, permeabilized and visualized for GFP expression, or stained with mouse monoclonal anti-phospho(p)-H2AX (Ser139) (pH2AX) (Millipore, Billerica, MA), rabbit polyclonal anti-RAD51 (Millipore), and rabbit polyclonal anti-cleaved caspase-3 (Cell Signaling Technology, Beverly, MA) as described [ 19 ].

Techniques: Expressing, Transfection, Staining

Journal: Gynecologic oncology

Article Title: Panobinostat sensitizes cyclin E high, homologous recombination-proficient ovarian cancer to olaparib

doi: 10.1016/j.ygyno.2016.07.088

Figure Lengend Snippet: A) IF analysis of the effects of panobinostat pre-treatment (25nM; 24h)/panobinostat (25nM; 24h) and olaparib (10µM) co-treatment combination regimen (24h) panobinostat (25nM)/olaparib (10µM) combination (24h) on pH2AX expression in OVCAR-3 cells. B) Representative images showing pH2AX staining (green) and DAPI-stained nuclei (blue). C) Western blot analysis pH2AX, cleaved PARP and cleaved caspase-3 expression in OVCAR-3 cells treated as above. Actin and total histone H3 were loading controls. D) IF analysis of cleaved caspase-3 expression in cells treated as above. Values for A) and D) are mean+SD for 3 independent experiments. At least 100 cells were counted (×40) for each drug treatment per experiment. *p<0.01 compared to vehicle; ap<0.01 relative to olaparib alone; bp<0.01 relative to panobinostat alone, all Student’s t test. E) Representative images of cleaved caspase-3 staining (green) and DAPI-stained nuclei (blue). Scale bars in B) are 20µm and in E) are 10µm.

Article Snippet: Immunofluorescence Following drug treatment and/or transient transfection with the HR reporter plasmid pDRGFP and endonuclease encoding pCBASce1 (I-Sce1) (both gifts from Maria Jasin; Addgene plasmids #26475 and #26477, respectively) [ 27 , 28 ] plasmids using Lipofectamine 2000 according to manufacturer’s instructions (Invitrogen Corp., Carlsbad, CA), ovarian cancer cells were fixed, permeabilized and visualized for GFP expression, or stained with mouse monoclonal anti-phospho(p)-H2AX (Ser139) (pH2AX) (Millipore, Billerica, MA), rabbit polyclonal anti-RAD51 (Millipore), and rabbit polyclonal anti-cleaved caspase-3 (Cell Signaling Technology, Beverly, MA) as described [ 19 ].

Techniques: Expressing, Staining, Western Blot

Journal: Gynecologic oncology

Article Title: Panobinostat sensitizes cyclin E high, homologous recombination-proficient ovarian cancer to olaparib

doi: 10.1016/j.ygyno.2016.07.088

Figure Lengend Snippet: A) Schematic of drug treatment experiment in nude mice injected subcutaneously with SKOV-3 cells. Mice (10 per group) were pre-treated for one week with vehicle or panobinostat, and then subsequently with vehicle, panobinostat and/or olaparib for three weeks as shown (IP: intraperitoneal; PO: per os, oral gavage). B) Time course measurements of tumor volume at weekly intervals (single arrow: start of panobinostat pre-treatment; double arrow: start of full drug regimen). C) Tumor weights at sacrifice. Tumors are shown in D). E) Western blot analysis of cyclin E, BRCA1, cleaved PARP, PCNA and pH2AX protein expression in harvested tumors. Actin and histone H3 were loading controls. F) Densitometry analysis of expression relative to corresponding actin or histone H3 levels. Values are mean+ SD; *p<0.01 single drug effect relative to vehicle; ap<0.01 combination drug effect relative to olaparib; bp<0.01 combination drug effect relative to panobinostat, Student’s t test.

Article Snippet: Immunofluorescence Following drug treatment and/or transient transfection with the HR reporter plasmid pDRGFP and endonuclease encoding pCBASce1 (I-Sce1) (both gifts from Maria Jasin; Addgene plasmids #26475 and #26477, respectively) [ 27 , 28 ] plasmids using Lipofectamine 2000 according to manufacturer’s instructions (Invitrogen Corp., Carlsbad, CA), ovarian cancer cells were fixed, permeabilized and visualized for GFP expression, or stained with mouse monoclonal anti-phospho(p)-H2AX (Ser139) (pH2AX) (Millipore, Billerica, MA), rabbit polyclonal anti-RAD51 (Millipore), and rabbit polyclonal anti-cleaved caspase-3 (Cell Signaling Technology, Beverly, MA) as described [ 19 ].

Techniques: Injection, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Alcohol-dysregulated microRNAs in hepatitis B virus-related hepatocellular carcinoma

doi: 10.1371/journal.pone.0178547

Figure Lengend Snippet: (A-B) MTS assay indicates that miR-944 and miR-223-3p knockdown sensitizes liver cells to doxorubicin, as shown by the reduction in doxorubicin IC-50. (C-D) Immunofluorescence images showing increased expression of γ-H2AX in cells treated with both doxorubicin and miRNA siRNAs compared to cell treated with doxorubicin alone. Cells were stained with anti-γH2AX (red) and DAPI (blue).

Article Snippet: Cells were subsequently treated with 0.25 μg/mL doxorubicin for 48 hours, and were fixed with 4% paraformaldehyde and blocked in goat serum at room temperature, followed by incubation with anti-phospho-Histone H2AX (JBW301) mouse monoclonal antibody (Cell Signaling Technology).

Techniques: MTS Assay, Immunofluorescence, Expressing, Staining