phospho histone h2ax  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Reciprocal links between methionine metabolism, DNA repair and therapy resistance in glioblastoma

doi: 10.1101/2024.11.20.624542

Figure Lengend Snippet: A-B). U87 cells or HF2303 spheres were grown in media with methionine or media depleted of methionine and irradiated (4 Gy). Cells or spheres were harvested at the indicated time points for γ-H2AX staining. Data is presented as mean±SEM for 3 biological replicates. C-D). U87 cells or HF2303 spheres were grown either untreated or treated with AGI- 41998, irradiated (4GY) and harvested for γ-H2AX staining at the indicated time points post-RT(4 Gy). Data is presented as mean±SEM for 3 biological replicates. E-F). U87 MTAP cells or HF2303 MTAP spheres were grown in media with methionine or media depleted of methionine and cells or spheres were harvested for γ-H2AX staining at the indicated time points post-RT(4 Gy). Data is presented as mean±SEM for 3 biological replicates. G-H). U87 MTAP cells or HF2303 MTAP spheres were untreated or treated with AGI-41998 and harvested for γ-H2AX staining at the indicated time points post- RT(4 Gy). Data is presented as mean±SEM for 3 biological replicates. I-J). Western blot showing the reduction of DNA repair proteins ATM, FANCA and FANCD2 in U87 cells and HF2303 neurospheres respectively. K-L). Western blot showing the levels of DNA repair proteins ATM, FANCA and FANCD2 in U87 MTAP cells and HF2303 MTAP neurospheres respectively. M-N). U87 cells and HF2303 neurospheres were treated with SAM overnight and retreated with SAM 1 hr before RT (4 Gy) and collected at indicated time points for γ-H2AX staining. Data is presented as mean±SEM for 3 biological replicates. N). P values were obtained in comparison with the control (DMSO). A-N). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001.

Article Snippet: Mouse monoclonal antibody anti-phospho- Histone H2AX (1:1000 dilution; Cat # 9718S, Cell Signaling) and goat anti-mouse IgG Alexa fluor 594 secondary antibody, (1:2000 dilution; Cat# A-11005, Invitrogen) were used for γ-H2AX foci staining.

Techniques: Irradiation, Staining, Western Blot, Comparison, Control

Journal: Protein Science : A Publication of the Protein Society

Article Title: Phosphorylation of cytochrome c at tyrosine 48 finely regulates its binding to the histone chaperone SET / TAF ‐Iβ in the nucleus

doi: 10.1002/pro.5213

Figure Lengend Snippet: Interaction of WT and Y48D cytochrome c with SET/TAF‐Iβ in the nucleus of MEF cells on basal DNA damage levels. (a) Immunofluorescence analyses of γ‐H2AX and C c in WT (endogenous C c ) and C c −/− (transfected or not with WT or Y48D C c species) MEF cells. The subcellular distribution of C c and γ‐H2AX was visualized using anti‐C c and anti‐γ‐H2AX antibodies—namely, FITC and Alexa Fluor 568, respectively. NT stands for non‐transfected cells but treated equally to the transfected and WT control cells, including incubation with both primary and secondary antibodies. Scale bars are 20 μm. (b) In situ PLA detection of C c ‐SET/TAF‐Iβ complexes and immunofluorescence labeling of C c in C c −/− MEF transfected with an empty vector, WT C c or Y48D C c . Representative projections of each condition with PLA spots in red and nuclei in blue are shown. Scale bars are 20 μm. (c) Quantitation of PLA spots per nucleus ( n = 27 for empty vector, n = 28 for WT C c , and n = 27 for Y48D C c ). Black lines and whiskers represent the mean and the SD, respectively. Statistical significance was calculated using the Mann–Whitney test (*** p < 0.001; “ns” stands for nonsignificant).

Article Snippet: For the detection of C c and γ‐H2AX, cells were then incubated with rabbit anti‐C c serum, obtained from male rabbits immunized with 20 μg mL −1 recombinant C c in 0.85% NaCl and with mouse anti‐phospho‐histone H2AX, clone JBW301 (Ser139, Sigma‐Aldrich, 05–636), both at a 1:200 dilution in blocking buffer overnight at 4°C, washed three times in PBS for 5 min and probed with goat anti‐rabbit IgG‐FITC (Sigma‐Aldrich, F9887) and goat anti‐mouse IgG Alexa Fluor 568 (Abcam, ab175473) antibodies, both 1:400 diluted in blocking buffer for 1 h at RT.

Techniques: Immunofluorescence, Transfection, Control, Incubation, In Situ, Labeling, Plasmid Preparation, Quantitation Assay, MANN-WHITNEY

Journal: EFSA Journal

Article Title: Re‐evaluation of saccharin and its sodium, potassium and calcium salts (E 954) as food additives

doi: 10.2903/j.efsa.2024.9044

Figure Lengend Snippet: Summary table of the in vitro genotoxicity studies on saccharins (E 954).

Article Snippet: The test is based on the quantification of the phosphorylated histone H2AX (γH2AX) in HepG2 cells using a high‐throughput technique based on Infrared Imaging Scanning , 24 h treatment in the absence of metabolic activation. Maximum tested concentration 1 mM (no other details provided) , Saccharin from Sigma‐Aldrich (purity not stated) , Negative , Reliable with restrictions , Limited/Limited , Khoury et al. ( ) .

Techniques: In Vitro, Concentration Assay, Ames Test, Activation Assay, Autoradiography, Single Cell Gel Electrophoresis, MTT Assay, High Throughput Screening Assay, Flow Cytometry, Mutagenesis, Screening Assay, Expressing, Control, Luciferase, Incubation, Imaging, High Content Screening, Fluorescence