Extended Data Fig. 1: Gel electrophoretic analysis and flow cytometric validation of GFP_Hx, mPlum_A-8oxoG and mOrange_8oxoG-C reporter plasmids.
From: Large-scale preparation of fluorescence multiplex host cell reactivation (FM-HCR) reporters
a, Analytical digest and flow cytometric validation of GFP_Hx plasmid. Lane 1: NEB 1-kb MW ladder; lane 2: pMax_GFP_C289T ccDNA; lane 3: HIA overnight extension reaction for GFP_Hx; lane 4: pMax_GFP_C289T ccDNA; lane 5: pMax_GFP_C289T after a 45-min ApaLI digestion at 37 °C, which cleaves two restriction sites, resulting in two linear DNA fragments; lane 6: GFP_Hx ccDNA after T5 Exo and PEG purification steps; and lane 7: GFP_Hx after a 45-min ApaLI digestion at 37 °C, in which the Hx lesion blocks ApaLI cleavage of one restriction site, leaving a single linearized fragment. At right: flow cytometric quantitation of percent reporter expression and normalized relative reporter expression in WT HAP cells compared to MPG−/− HAP cells. b, Analytical digestion and flow cytometric validation of mPlum_A-8oxoG plasmid. Lane 1: HIA overnight extension reaction for mPlum_A-8oxoG; lane 2: pMax_mPlum ccDNA; lane 3: pMax_mPlum after a 45-min Fpg endonuclease digestion at 37 °C; lane 4: mPlum_A-8oxoG after T5 Exo and PEG purification steps; lane 5: mPlum_A-8oxoG after a 45-min Fpg endonuclease digestion at 37 °C, resulting in plasmid nicking and upward mobility shift. At right: flow cytometric quantitation of percent reporter expression and normalized relative reporter expression in WT HAP cells compared to MUTYH−/− HAP cells. c, Analytical digestion and flow cytometric validation of mOrange_8oxoG-C plasmid. Lane 1: NEB 1-kb MW ladder; lane 2: pMax_mOrange_A215C ssDNA; lane 3: pMax_mOrange_A215C ocDNA; lane 4: pMax_mOrange_A215C ccDNA; lane 5: pMax_mOrange_A215C after a 45-min Fpg endonuclease digestion at 37 °C; lane 6: mOrange_8oxoG-C ccDNA; and lane 7: mOrange_8oxoG-C after a 45-min Fpg endonuclease digestion at 37 °C, which introduces a nick at the 8oxoG lesion, resulting in upward mobility shift. At right: flow cytometric quantitation of percent reporter expression and normalized relative reporter expression in WT MEF cells compared to OGG1−/− MEF cells. Error bars represent s.e.m. from three to four biological replicates; differences of statistical significance (*, P < 0.05; ***, P < 0.005; ****, P < 0.0001) were determined by unpaired two-tailed t test.
