γ h2a x phospho s139

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γ-H2A.X (phospho S139) is a laboratory product that detects the phosphorylation of serine 139 on the histone variant H2A.X. This phosphorylation is a well-established marker of DNA double-strand breaks.

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γ-H2A.X(S139 (5 citations)

3 protocols using γ h2a x phospho s139

Cell Line Characterization and Antibody Validation

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The HeLa cell line (BCRC-60005), C33A cell line (BCRC-60554), DLD-1 cell line (BCRC-60132), LS174T (BCRC-60053), HA22T (BCRC-60168), and HA59T (BCRC-60169) were purchased from the Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan. HeLa, C33A, and LS174T Cells were maintained in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100-U/mL streptomycin, and 100-mg/ml penicillin. The DLD-1 cells were maintained in Roswell Park Memorial Institute medium supplemented with 10% FBS, 100-U/mL streptomycin, and 100-mg/ml penicillin. HA22T and HA59T were maintained in MEM supplemented with 10% FBS, 100-U/mL streptomycin, and 100-mg/ml penicillin (all from Gibco). Primary antibodies targeting the following proteins were used: CDK1, CDK2, cyclin A, cyclin B, cyclin E, p53, and corresponding secondary antibodies (goat anti-rabbit IgG and goat anti-mouse IgG) (all from Santa Cruz); β-actin, ROGDI, p21, γ-H2A.X (phospho S140) and γ-H2A.X (phospho S139) (all from Abcam); and ROGDI (Proteintech, 17047-1-AP).

Chen Y.F., Cho J.J., Huang T.H., Tseng C.N., Huang E.Y, & Cho C.L. (2016). Downregulation of a novel human gene, ROGDI, increases radiosensitivity in cervical cancer cells. Cancer Biology & Therapy, 17(10), 1070-1078.

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Immunohistological Analysis of Explants

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For immunohistological analysis, explants attached to Millicell‐CM culture plate inserts were fixed overnight with 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 3 μm. Sections were stained with rabbit polyclonal antibodies to cleaved caspase 3 (Cell Signaling Technology, Danvers, MA), to Ki67 (clone SP‐6, Thermo Fisher Scientific), and to γH2AX (phospho‐S139; Abcam, Cambridge, UK) or with a mouse monoclonal antibody to phosphorylated histone H3 (Cell Signaling Technology). Immune complexes were detected as described previously 18. Quantifications were performed with ImageJ.

Minami N., Maeda Y., Shibao S., Arima Y., Ohka F., Kondo Y., Maruyama K., Kusuhara M., Sasayama T., Kohmura E., Saya H, & Sampetrean O. (2017). Organotypic brain explant culture as a drug evaluation system for malignant brain tumors. Cancer Medicine, 6(11), 2635-2645.

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Inhibition of PLK1 and DNA Damage Response Pathways

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Antibodies were as follows: p53 (DO-1; Moravian Biotechnology), p21 ((H-164): sc-756; SantaCruz Biotechnology), PLK1 (208G4; Cell Signaling Technology), actin (A2066; Sigma), Poly (ADP-Ribose) Polymerase-1 (PARP) (9542; Cell Signaling Technology), γ-H2AX (Phospho-S139) (ab11174; AbCam), p53 (Phospho-S15) (#9284; Cell Signaling Technology), MDM2 (4B2; Moravian Biotechnology), Gamma-tubulin (T65557; Sigma), Histone H3 ((D1H2): 4499; Cell Signaling Technology), Histone H3 (Phospho-S10) (06–570; Millipore), BrdU Pure ((B44): 347580; Becton Dickinson).
PLK1 inhibitors, GSK461364 and BI6727, ATM and ATR inhibitors, KU-55933 and VE-821, and etoposide were from Selleckchem. The specificity and efficacy of GSK461364 and BI6727 have been described previously^{36 (link),37 (link)}. S-Trityl-L-cysteine (STLC) was from Sigma. Taxol was from LC Labs. Nocodazole was from Millipore.

Smith L., Farzan R., Ali S., Buluwela L., Saurin A.T, & Meek D.W. (2017). The responses of cancer cells to PLK1 inhibitors reveal a novel protective role for p53 in maintaining centrosome separation. Scientific Reports, 7, 16115.

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