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. 2019 May 25;47(14):7418–7429. doi: 10.1093/nar/gkz466

Figure 2.

Figure 2.

PARP limits local AID mediated DNA damage induction. (A) Schematic of the F3H experimental system used to localize AID-ΔNES-GFP to a defined nuclear structure. GFP fusion proteins are tethered to a lac array in U2OS 2-6-3 cells by a GFP-binding protein-LacI fusion, and the DNA damage caused is detected by γH2AX or 53BP1 staining. (B) Representative confocal images of U2OS 2-6-3 cells expressing either GFP alone or AID-ΔNES-GFP treated with DMSO, 1 μM Olaparib or 1 mM 3-AB for 24 h. DNA damage induction via AID was visualized using antibodies against γH2AX and 53BP1. (C and D) Percentage of cells displaying co-localization of AID-ΔNES-GFP or GFP with γH2AX (C) and 53BP1 (D), respectively. Data from three independent experiments are shown. (E and F) Scatter plot of fluorescence intensities of γH2AX (E) and 53BP1 (F) at the Lac operator array in 2-6-3 cells expressing AID-ΔNES-GFP or GFP. Data from >60 cells gathered in three independent experiments are shown. Statistical significance was determined using the Student's t-test (*P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001). Error bars indicate the standard deviation.