Journal: FEBS Open Bio
Article Title: TRIM29 is required for efficient recruitment of 53BP1 in response to DNA double‐strand breaks in vertebrate cells
Figure Lengend Snippet: Clonogenic survival assays after genotoxic treatments. Clonogenic survival assays of WT , TRIM29 −/−/−/+ , Ku70 −/− , PALB2 −/− , BRCA1 −/− , and REV1 −/− cells against etoposide (A), camptothecin (B), cisplatin (C), olaparib (D), and UV‐C (E) treatments. Data are the mean ± SD of three independent experiments ( * P ≤ 0.05; ** P ≤ 0.01, percent survival of WT cells versus TRIM29 −/−/−/+ cells, Student’s t ‐test).
Article Snippet: Cells were harvested onto the surface of a glass slide at the indicated time points using a Cytospin4 cytocentrifuge (Thermo Fisher Scientific), fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X‐100 for 10 min, and blocked in Odyssey® blocking buffer (LI‐COR Biosciences, Lincoln, NE, USA) at room temperature for 1 h. The cells were then incubated with mouse monoclonal anti‐ɣ‐H2AX, Ser 139 antibody (1:500; CST, cat. #80312, Danvers, MA, USA), and rabbit polyclonal anti‐53BP1 antibody (1:500; Novus Biologicals, cat. #NB100‐904, Centennial, CO, USA) for the etoposide‐induced foci formation assay, or rabbit monoclonal anti‐ɣ‐H2AX, Ser 139 antibody (1:1000; CST, cat. #9718) and mouse monoclonal anti‐RAD51 antibody (1:200; Santa Cruz, cat. #sc‐398587, Dallas, TX, USA) for the camptothecin‐induced foci formation assay at room temperature for 1 h. After intensive washing, the cells were incubated with goat anti‐mouse IgG conjugated with Dylight 594 (1:500; Thermo Fisher Scientific, cat. #35510) and goat anti‐rabbit IgG conjugated with Dylight 488 (1:500; Thermo Fisher Scientific, cat. #35552), and donkey anti‐rabbit IgG conjugated with Alexa Fluor 594 (1:500; Thermo Fisher Scientific, cat. #A‐21207) and donkey anti‐mouse IgG conjugated with Alexa Fluor 488 (1:500; Thermo Fisher Scientific, cat. #A‐21202) for etoposide‐ and camptothecin‐induced foci formation assays, respectively, at room temperature for 1 h. Cells were counterstained with Hoechst 33258 (1:2000; Thermo Fisher Scientific), and foci of ɣ‐H2AX, 53BP1, and RAD51 were observed under a fluorescence microscope from Nikon (Eclipse Ci Series, Tokyo, Japan).