PTEN-mediated DNA Damage Response

HCT116 PTEN-null cells were seeded on Lab-Tek eight-well chamber coated with laminin (L2020, Sigma) and incubated overnight. Cells were treated with 1 mM hydroxyurea (H8627, Sigma) for 16 h after a 24-h transfection with either wild-type PTEN or mutated constructs. Cells were fixed as previously described16 (link) and immunolabeled with a rabbit anti-phosphorylated histone H2AX (γH2AX) antibody (ab2893, Abcam) and a anti-rabbit IgG Alexa 568 antibody (Invitrogen). Images were acquired by confocal fluorescence microscopy (Zeiss LSM 780 NLO) equipped with a 63× objective. The numbers of nuclear foci of phosphorylated histone H2AX were manually counted. Nuclei were identified by staining with Hoechst 33258 (Molecular Probes).

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Nguyen H.N., Yang J.M., Miyamoto T., Itoh K., Rho E., Zhang Q., Inoue T., Devreotes P.N., Sesaki H, & Iijima M. (2015). Opening the conformation is a master switch for the dual localization and phosphatase activity of PTEN. Scientific Reports, 5, 12600.

Publication 2015

hydroxyurea ab2893 LSM 780 NLO Hoechst 33258

Alexa 568 Anti igg Antibody Cells Fluorescence microscopy Hct116 cells Histone h2ax Hoechst 33258 Hydroxyurea Laminin Molecular probes Nuclei Pten Rabbit Transfection

Corresponding Organization :

Other organizations : Johns Hopkins University, Johns Hopkins Medicine

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Variable analysis

independent variables

PTEN construct (wild-type or mutated)

dependent variables

Number of nuclear foci of phosphorylated histone H2AX

control variables

HCT116 PTEN-null cells
Lab-Tek eight-well chamber coated with laminin
Incubation duration (overnight)
Hydroxyurea treatment (1 mM for 16 h)
Transfection duration (24 h)
Fixation method
Immunolabeling with anti-phosphorylated histone H2AX (γH2AX) antibody and anti-rabbit IgG Alexa 568 antibody
Confocal fluorescence microscopy (Zeiss LSM 780 NLO, 63× objective)
Nuclei staining with Hoechst 33258

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