Journal: Cells

Article Title: The Ubiquitin Ligase RNF138 Cooperates with CtIP to Stimulate Resection of Complex DNA Double-Strand Breaks in Human G1-Phase Cells

doi: 10.3390/cells11162561

Figure Lengend Snippet: RNF138 is required for the recruitment of CtIP to DSBs in G1-phase cells. NFFhTERT, HeLa.S-Fucci, or U2OS cells, the latter ectopically expressing CtIP-mCherry, RNF138 proficient or deficient [mock KD and RNF138 KD (see ) or wt and RNF138 KO, respectively] were irradiated in a low angle (5 × 10 6 p./cm 2 ) with carbon ions (6.5 MeV/nucleon) or low-energy iron ions and fixed 1 h after irradiation. The samples were immunofluorescence-stained for CtIP and the cell-cycle marker Geminin (Geminin positive: S and G2 cells, Geminin negative: G1 cells; NFFhTERT and U2OS only) or for CtIP and the DSB marker γH2AX (HeLa.S-Fucci cells). DNA was counterstained with DAPI to visualize the nuclei of all cells. Cells positive for CtIP-decorated ion traversals (streak-like pattern) within their nuclei were considered CtIP positive. ( a ) At least 100 cells per condition were analyzed from at least 6 fields of view in order to average the number of CtIP-positive cells. n = 1, error = SEM. Two-tailed Mann-Whitney test, p values: * p < 0.05; ** p < 0.01; **** p < 0.0001. ( b ) Representative image of CtIP signal in WT and RNF138 KO HeLa.S-Fucci cells. The CtIP signal is overlaid by the signal of mAG-tagged Geminin, a part of the Fucci system. mKO2-tagged Cdt1 of the Fucci system served as cell-cycle marker. White arrows indicate CtIP-positive cells. An enlarged version of the areas highlighted by dashed boxes is shown to the right.

Article Snippet: Primary antibodies for immunofluorescence staining were diluted in 1x PBS, 0.4% BSA: α53BP1 (Ab-1) (rabbit, Calbiochem, PC712, 1:500), αCENP-F (rabbit, Novus, NB500-101, 1:750), αCtIP (rabbit, Bethyl Laboratories, A300-488A, 1:100), αGeminin (mouse, clone 1A8, Novus Biologicals, H00051053-M01, 1:100), αγH2AX (Ser 139) (mouse, clone JBW301, Millipore, 05-636, 1:500), αKu80 (mouse, clone 111, Thermo Scientific, MA5-12933, 1:100), αRPA/P34 (mouse, clone 9H8, Sigma, R1280, 1:3000).

Techniques: Expressing, Irradiation, Immunofluorescence, Staining, Marker, Two Tailed Test, MANN-WHITNEY

Journal:

Article Title: A requirement for replication in activation of the ATR-dependent DNA damage checkpoint

doi: 10.1101/gad.1013502

Figure Lengend Snippet: Geminin blocks UV-induced accumulation of checkpoint and replication proteins on chromatin. (A) Binding of xATR, xRad1, and xORC2 to chromatin in interphase extract was assayed after mock, UV, or aphidicolin (APH) treatment. Chromatin (2000/μL) was isolated at the given time points, and chromatin-bound proteins were analyzed by Western blotting with antibodies to xATR, xRad1, or xORC2. (B) Chromatin binding of the p180 subunit of xPolα, xRPA70, and xORC2 was assayed after mock, UV, and aphidicolin treatment by Western blotting. Samples were treated and prepared as described in A. (C) Interphase extract was pretreated for 10 min with geminin (+gem) before addition of mock- or UV-treated chromatin (2000/μL). Chromatin was isolated after 100 min, and chromatin-bound proteins were analyzed as described above.

Article Snippet: Production of antibodies, recombinant geminin Rabbit polyclonal xRad1 and xATR antibodies were raised at a commercial facility (Josman, Napa, CA).

Techniques: Binding Assay, Isolation, Western Blot