ej5 gfp nhej reporter addgene  (Addgene inc)

Journal: The Journal of biological chemistry

Article Title: PC4-mediated Ku complex PARylation facilitates NHEJ-dependent DNA damage repair.

doi: 10.1016/j.jbc.2023.105032

Figure Lengend Snippet: Figure 6. PC4-mediated XRCC6 PARylation promotes XRCC6 efficient loading on DSB sites. A, representative time-lapse images showing the recruitment of XRCC6-GFP to multiphoton tracks in Huh7 cells with or without PC4 knockdown. Scale bar, 10 μm. B, quantification of XRCC6-GFP fluo- rescence intensity at DNA damage sites in A. Data were derived from three independent experiments. In each experiment, 50 different cells were investigated. C, quantification of cell death in XRCC6-GFP cells upon laser microradiation with or without PC4 knockdown. n = 3. D, quantification of XRCC6- GFP fluorescence intensity at DNA damage sites in NC, siPC4, siPARP1, or siPC4+siPARP1 group. Data were derived from three independent experiments. E, characterization of the NHEJ repair efficiency by EJ5-GFP and FACS in NC, siPC4, siPARP1, or siPC4+siPARP1 group. n = 3. F, quantification of tail moments in NC, siPC4, siPARP1, or siPC4+siPARP1 group as determined by a neutral comet assay. n = 3. G, the responses of survival factions of Huh7 cells to X-ray irradiation in NC, shPC4, siXRCC6, or shPC4+siXRCC6 group. n = 3. All graphed data were shown as means ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001. DSBs, double-strand breaks; FACS, fluorescent activated cell sorting; NHEJ, nonhomologous end joining; PC4, human positive cofactor 4.

Article Snippet: NHEJ reporter plasmid EJ5-GFP (Addgene plasmid #44026) or HR reporter plasmid DR-GFP (Addgene plasmid #26475) was transfected into Huh7 cells respectively.

Techniques: Knockdown, Derivative Assay, Neutral Comet Assay, Irradiation, FACS