Journal: Scientific Reports

Article Title: Role of areca nut induced JNK/ATF2/Jun axis in the activation of TGF-β pathway in precancerous Oral Submucous Fibrosis

doi: 10.1038/srep34314

Figure Lengend Snippet: ( a ) Immunoblot representing compromise in areca nut induced pJNK levels in the presence of muscarinic acid receptor inhibitor (atropine) at 0.5 hours. ( b ) Representative images from depicting areca nut induced calcium release. White arrows depict the cells with higher calcium levels post areca nut treatment. Magnification factor; 10X, Scale bar = 100 μm. ( c ) Immunoblot representing compromise in areca nut induced pCAMKII levels in the presence of muscarinic acid receptor inhibitor (atropine) at 0.5 hours. ( d ) Immunoblot representing compromise in areca nut induced pJNK levels in the presence of CAMKII inhibitor (KN93) at 0.5 hours. ( e ) Representative images for live confocal imaging of areca nut induced ROS at 0 and 30 minutes. Magnification factor; 63X. ( f ) Immunoblot representing compromise in areca nut induced pJNK levels in the presence of ROS inhibitor (DPI) at 0.5 hours. β-actin is used as loading control in all the immunoblots.

Article Snippet: Dual staining for pATF2 and p-c-Jun was detected by Zeiss LSM880Airyscan Superresolution microscopy (Magnification factor: 63X; Zoom: 1.8; Sale bar: 10 μm).

Techniques: Western Blot, Imaging

Journal: Scientific Reports

Article Title: Role of areca nut induced JNK/ATF2/Jun axis in the activation of TGF-β pathway in precancerous Oral Submucous Fibrosis

doi: 10.1038/srep34314

Figure Lengend Snippet: ( a ) Representative immunofluorescence images of areca nut induced nuclear localized pATF2 and p-c-Jun at 2 hours. Magnification: 63X; Zoom: 1.8; Scale bar = 10 μm. ( b ) Bar graph of qPCR results of chIP assay showing fold enrichment of pATF2 and p-c-Jun on TGF-β2 promoter at 2 hours upon areca nut treatment compared to untreated cells. ( c ) Bar graph showing fold change at 2 hour of areca nut induced TGF-β2 and its compromise upon transient knock down of ATF2 or c-Jun. ( d,e ) Immunoblots representing pSMAD2 levels at 2 hour areca nut treatment and its compromise upon transient knock down of ATF2 or c-Jun. β-actin is used as loading control in all the immunoblots. *** , ** represent p values ≤ 0.0001; 0.001 respectively. (5H; 5 μg/ml areca nut extract).

Article Snippet: Dual staining for pATF2 and p-c-Jun was detected by Zeiss LSM880Airyscan Superresolution microscopy (Magnification factor: 63X; Zoom: 1.8; Sale bar: 10 μm).

Techniques: Immunofluorescence, Western Blot

Journal: Radiation Research

Article Title: Specific ATM-Mediated Phosphorylation Dependent on Radiation Quality

doi: 10.1667/rr1354.1

Figure Lengend Snippet: FIG. 1. Radiation induces DSBs that then initiate a complex cascade of phosphorylations. Phosphorylations on these proteins are important in the resolution and repair of DNA damage after irradiation. The phospho- proteins studied are shown in the white ovals, proteins involved in phos- phorylation of these proteins are shown in the gray symbols, and the processes the proteins are believed to play a role in are in boxes. Phos- phorylation of H2AX on Ser 139 is mediated by ATM, ATR and DNA- PKcs. ATM-dependent phosphorylations occur on ATF2 and SMC1 at Ser 490/498 and Ser 957, respectively. pATF2 and pSMC1 are proposed to have roles both in DNA repair and maintaining S-phase block after damage. The star symbols (*) represent phosphorylations occurring on these proteins, and arrows indicate proteins involved in phosphorylating these proteins. ATF2 has also been shown to have a role in increasing activation of ATM after damage: thus the dual arrow between these pro- teins.

Article Snippet: Antibodies Primary antibodies used included mouse monoclonal -H2AX (1:800 dilution), pSMC1 Ser957 (1:800 dilution) from Upstate (Lake Placid, NY), and rabbit polyclonal pATF2 Ser490/498 (1:1000 dilution) from Rockland (Gilbertsville, PA).

Techniques: Irradiation, Blocking Assay, Activation Assay

Journal: Radiation Research

Article Title: Specific ATM-Mediated Phosphorylation Dependent on Radiation Quality

doi: 10.1667/rr1354.1

Figure Lengend Snippet: FIG. 3. Comparison of phospho-protein profiles after exposure of cells to X rays and iron ions using flow cytometry. Median values of total fluorescence were obtained for each dose and time. Average median values from three independent experiments are plotted relative to the control for both X rays (top panels) and iron ions (lower panels), with error bars representing standard errors. Each time is represented by a different symbol and color (blue 0.5 h, red 2 h, green 4 h, aqua 8 h, purple 24 h). The kinetics of -H2AX (panel A), pATF2 (panel B) and pSMC1 (panel C) after X-ray exposure and -H2AX (panel D), pATF2 (panel E) and pSMC1 (panel F) after iron-ion exposure are shown.

Article Snippet: Antibodies Primary antibodies used included mouse monoclonal -H2AX (1:800 dilution), pSMC1 Ser957 (1:800 dilution) from Upstate (Lake Placid, NY), and rabbit polyclonal pATF2 Ser490/498 (1:1000 dilution) from Rockland (Gilbertsville, PA).

Techniques: Comparison, Cytometry, Control

Journal: Radiation Research

Article Title: Specific ATM-Mediated Phosphorylation Dependent on Radiation Quality

doi: 10.1667/rr1354.1

Figure Lengend Snippet: FIG. 4. Comparison of phospho-protein profiles after exposure of cells to low doses (0.05 to 0.5 Gy) of X rays and iron ions using flow cytometry. Data are shown only in this lower dose range for each time to better exhibit differences. Median values are plotted relative to the control levels for both X rays and iron ions, and error bars represent standard errors. Data are from three independent iron-ion and X-ray experiments. Each time is represented by a different symbol and color (blue 0.5 h, red 2 h, green 4 h, aqua 8 h, purple 24 h). The kinetics of -H2AX (panel A), pATF2 (panel B) and pSMC1 (panel C) after X-ray exposure and -H2AX (panel D), pATF2 (panel E) and pSMC1 (panel F) after iron-ion exposure are shown.

Article Snippet: Antibodies Primary antibodies used included mouse monoclonal -H2AX (1:800 dilution), pSMC1 Ser957 (1:800 dilution) from Upstate (Lake Placid, NY), and rabbit polyclonal pATF2 Ser490/498 (1:1000 dilution) from Rockland (Gilbertsville, PA).

Techniques: Comparison, Cytometry, Control

Journal: Radiation Research

Article Title: Specific ATM-Mediated Phosphorylation Dependent on Radiation Quality

doi: 10.1667/rr1354.1

Figure Lengend Snippet: FIG. 6. The percentages of cells showing various increases in phosphorylation over control levels of -H2AX (black bars) and pATF2 (white bars) after X-ray (left panel) and iron-ion (right panel) exposure. Error bars represent standard errors from three independent X-ray or iron-ion experiments.

Article Snippet: Antibodies Primary antibodies used included mouse monoclonal -H2AX (1:800 dilution), pSMC1 Ser957 (1:800 dilution) from Upstate (Lake Placid, NY), and rabbit polyclonal pATF2 Ser490/498 (1:1000 dilution) from Rockland (Gilbertsville, PA).

Techniques: Phospho-proteomics, Control

Journal: Radiation Research

Article Title: Specific ATM-Mediated Phosphorylation Dependent on Radiation Quality

doi: 10.1667/rr1354.1

Figure Lengend Snippet: FIG. 5. Comparison of radiation-induced -H2AX (panels A, D), pATF2 (panels B, E) and pSMC1 (panels C, F) foci from a representative X-ray experiment with those (top panels) from a representative iron-ion (bottom panels) experiment. Images of microscopic foci detected 2 h after a 0.5-Gy exposure to X rays (top panels) or iron ions (lower panels) are shown. Each phospho-site antibody was indirectly labeled with Molecular Probes 488 secondary antibody (green), and cells were counterstained with DAPI (blue). All images were taken with the same exposure times. White ovals outline the DAPI-stained cells within each field that do not contain foci.

Article Snippet: Antibodies Primary antibodies used included mouse monoclonal -H2AX (1:800 dilution), pSMC1 Ser957 (1:800 dilution) from Upstate (Lake Placid, NY), and rabbit polyclonal pATF2 Ser490/498 (1:1000 dilution) from Rockland (Gilbertsville, PA).

Techniques: Comparison, Labeling, Staining

Journal: Radiation Research

Article Title: Specific ATM-Mediated Phosphorylation Dependent on Radiation Quality

doi: 10.1667/rr1354.1

Figure Lengend Snippet: FIG. 7. Comparison of the fluorescence levels of each phospho-protein at various times after X-ray (top panels) or iron-ion (lower panels) exposure. -H2AX phosphorylation levels are shown in dark blue, pATF2 in green, and pSMC1 in aqua. Levels are shown after a dose of 2 Gy (panels A, C) and 0.5 Gy (panels B, D) of X rays or iron ions. Data are shown as relative fluorescence for three independent iron-ion and X-ray experiments.

Article Snippet: Antibodies Primary antibodies used included mouse monoclonal -H2AX (1:800 dilution), pSMC1 Ser957 (1:800 dilution) from Upstate (Lake Placid, NY), and rabbit polyclonal pATF2 Ser490/498 (1:1000 dilution) from Rockland (Gilbertsville, PA).

Techniques: Comparison, Phospho-proteomics

Journal: Radiation Research

Article Title: Specific ATM-Mediated Phosphorylation Dependent on Radiation Quality

doi: 10.1667/rr1354.1

Figure Lengend Snippet: FIG. 8. Comparison of the fluorescence levels for each phospho-protein at various times after X-ray (top panels) or iron-ion (lower panels) exposure. -H2AX phosphorylation levels are shown in dark blue, pATF2 in green, and pSMC1 in aqua. Levels are shown after a dose of 0.1 Gy (panels A, C) and 0.05 Gy (panels B, D) of X rays or iron ions. Data are shown as relative fluorescence for three independent iron-ion and X-ray experiments.

Article Snippet: Antibodies Primary antibodies used included mouse monoclonal -H2AX (1:800 dilution), pSMC1 Ser957 (1:800 dilution) from Upstate (Lake Placid, NY), and rabbit polyclonal pATF2 Ser490/498 (1:1000 dilution) from Rockland (Gilbertsville, PA).

Techniques: Comparison, Phospho-proteomics

Journal: Radiation Research

Article Title: Specific ATM-Mediated Phosphorylation Dependent on Radiation Quality

doi: 10.1667/rr1354.1

Figure Lengend Snippet: FIG. 9. The relationship between phospho-protein kinetics and the cell cycle. Bars show the percentage of induced phosphorylation remaining in G1 (panels A and D), S (panels B and E) and G2 (panels C and F) phases of the cell cycle after a 2-Gy dose of X rays (top panels) or iron ions (lower panels). Levels of phosphorylated -H2AX (black bar), pATF2 (white bar), and pPSMC1 (gray bar) are shown. Bars represent standard errors for three independent iron-ion and X-ray experiments.

Article Snippet: Antibodies Primary antibodies used included mouse monoclonal -H2AX (1:800 dilution), pSMC1 Ser957 (1:800 dilution) from Upstate (Lake Placid, NY), and rabbit polyclonal pATF2 Ser490/498 (1:1000 dilution) from Rockland (Gilbertsville, PA).

Techniques: Phospho-proteomics