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. 2015 Mar 19;10(3):e0121581. doi: 10.1371/journal.pone.0121581

Fig 6. Analysis of NHEJ in MM cells.

Fig 6

(A) Map of pEGFP-Pem1-Ad2 modified from reference [36]. (B) Dot plots of nontransfected LINF903 cells (panel 1), LINF903 cells transfected with 2 μg pDSRed2-N1 (panel 2), with 0.5 μg of pEGFP-Pem1 (panel 3), or with both plasmids together (panel 4). Numbers of green and red cells were determined 24h after transfection by FACS. (C) Dot plots of LINF903 and U266 cell lines transfected with 0.5 μg of pEGFP-Pem1 or 0.5 μg of HindIII-digested pEGFP-Pem1-Ad2 plasmid, together with 2 μg of pDSRed2-N1. Total represented events were adjusted to correct for differences in transfection efficiencies, and same numbers of cells transfected with circular and/or control pDSRed2-N1 are shown (6,000 cells). These numbers of events were then represented in panels corresponding to transfections with digested molecules. (D) Percentage of NHEJ of HindIII- or SceI-digested plasmid in different cell lines. Mean of a minimum of three independent experiments is shown. (** p<0.01, * p<0.05, compared to LINF cells). (E) NHEJ efficiency in LINF, JJN3 and U266 cell lines carrying the integrated NHEJ reporter cassette. Stable pools were transfected with 5 μg of I-SceI endonuclease-expressing plasmid and 2 μg pDSRed2-N1 to correct for differences in transfection efficiencies. GFP+ and DsRed+ cells 24h after transfection are shown in panel 1 (A total of 6,000 GFP+ and/or red+ cells are shown). NHEJ efficiency was calculated as the ratio of GFP+/DsRed+ cells (panel 2). Data are the mean of three independent experiments (** p<0.01, compared to LINF cells). (F) Agarose gel showing PCR products of misrepaired products (1–14). The size of the PCR product from a correctly repaired DSB (c) is 628 bp. (G) Percentage of large deletions (≥20 bp, panel 1) and percentage of misrepaired plasmids using sequence microhomology (≥2 bp, panel 2) in LINF, U266, JJN3, and MM1S cell lines. (H) Deletion size (panel 1) and percentage of microhomology (panel 2) in plasmids recovered from U266 cells treated or not with mirin (100 μM) and NU1025 (50 μM). Cells were pretreated with the chemical inhibitors for 6h, transfected with EcoRI-digested plasmid, and cultured for 18h in the presence of the chemical inhibitors. (I) NHEJ activity remaining after inhibition of DNA-PKcs. Cells were transfected as in (C), and grown in the presence of 10 μM of NU7026.