Computational protocol: Unscheduled expression of CDC25B in S-phase leads to replicative stress and DNA damage

Similar protocols

Protocol publication

[…] Mouse anti-phospho Ser139 γ-H2AX (clone JBW 301, Upstate Biotechnology, Lake Placid, NY), rabbit anti-phospho Ser139 γ-H2AX (Upstate Biotechnology), mouse anti-Ha tag (clone Ha.11 Covance), rabbit anti-phospho H3-Ser210 (Upstate Biotechnology), rat anti-BrdU (clone BU1/75 Serotec), mouse anti-BrdU (Becton Dickinson), rabbit CDC25B antibody (C-20. Santa Cruz, CA), mouse anti-actin (Chemicon, Temecula, CA), rabbit anti-CDC45 (ref. 20685. Santa Cruz). Mouse rabbit and rat anti-IgG Alexa 488 and 594 for immunofluorescence (Molecular Probes, Invitrogen), rabbit and mouse anti-HRP antibodies (Cell Signalling).Cells cultured on glass coverslips were processed as previously described then incubated with rabbit anti-γ-H2AX and mouse anti-Ha tag or rabbit anti-phospho H3 Ser210 and mouse anti-phospho γ-H2AX followed by rabbit and mouse Alexa secondary antibody staining []. Cells were mounted in Vectashield anti-fade mounting medium and visualized using a DM6000 microscope (Leica, Wetzlar, Germany). For BrdU staining, cells were incubated with 30 μM BrdU (Calbiochem) for 15 min and fixed with 3.7% formaldehyde for 10 min. The cells were processed as described in [] with some modifications: they were washed with PBS and incubated with methanol for 5 min at -20°C then treated with PBS/0.5%Triton ×100/0.02% SDS for 30 min at room temperature. DNA was denatured using freshly prepared 1.5 M HCl, then neutralized by washing with 0.1 M sodium borate (pH 8.5) and PBS. To block non-specific binding, cells were incubated in 5%PBS-BSA, 30 min to overnight at 4°C then submitted to anti-γ-H2AX or anti-BrdU for 1 h then two washes with PBS followed by mouse anti-IgG Alexa 594 and rat anti-IgG Alexa 488 respectively.Replication focus detection with CldU and IdU was performed on U2OS or HCT116 cells blocked by thymidine (2.5 mM) for 17 h then released in DMEM for two hours. Cells were incubated in medium containing 100 μM CldU (Sigma, St Quentin, France) for 30 min then 100 μM IdU for the last 30 minutes after washing with hot medium. IdU incorporation was stopped with medium containing thymidine (1 mM) then cells were fixed with cold 70% ethanol. They were treated with 100% methanol at -20°C for 5 min, washed twice with PBS then incubated in 1.5 M HCl for 20 min. After two washes with PBS, they were incubated in 0.5% Tween20/0.25%BSA/5% fetal veal serum/PBS/(TBS) for 30 min in a humid box. Incubation in the primary antibody rat anti-BrdU against CldU and mouse anti-BrdU against IdU in TBS for 2 hours was followed by anti-rat IgG Alexa 594 and anti-mouse IgG Alexa 488 in TBS respectively. Cells were washed twice in 0.5% Tween/PBS then mounted in Vectashield solution and visualized using a DM 6000 microscope. Pictures were acquired with MetaMorph software, keeping the same intensities for each fluorescent dye for all the pictures of the same assay and the signals were measured using ImageJ software. IdU-CldU colocalization was quantified from the merge picture by dividing the colocalization area by the total area for each nucleus and the non-parametric Welch T corrected test was used to analyse the data. […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Application Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Neoplasms