Figure 6.

Suppression of in vivo MMEJ after Mirin-treatment or knocking down Mre11. (A) Schematic representation of the pMMEJ plasmid used to assess in vivo MMEJ repair. The pMMEJ plasmid was derived from the pEGFP-C3 plasmid by an insertion of 35 bp within the ORF of the wtEGFP gene. This creates an I-SceI megaendonuclease recognition site flanked by two 5 bp microhomologies. The repair of a linearized pMMEJ plasmid by MMEJ reconstitutes the wtEGFP gene and allows expression of EGFP. (B) WI-38VA13 (CF) and AT5BIVA (AT) cells were transfected with either linear or circular pMMEJ and with a mCherry transfection control plasmid. Repair was allowed and cells were analyzed for fluorescent protein expression by flow cytometry. Cells treated with 25 mM Mirin or in which Mre11 was knocked down by transduction with lentivirus encoding Mre11 shRNA (Mre11 sh3 or Mre11 sh4) were also analyzed. Cells transduced with empty vector or virus-free transduction medium were included as controls. Repair through MMEJ is represented by %EGFP expression; %EGFP expression = (number of cells expressing both EGFP and mCherry/number of cells expressing mCherry) × 100.