Fig 1: MMS generates DSB in a BER-dependent manner.A, U2OS cells were used for immunofluorescent microscopy using DAPI (nuclei staining), γH2AX (AF488), and 53BP1 (AF594) after exposing cells to one, two, and three hours of 3mM MMS respectively.B, Overlapping/colocalizing γH2AX-53BP1 foci were quantified on a per nuclei basis using n=50 cells.C, γH2AX foci per cell were quantified for n=50 cells after MMS exposure.D, Comet assay under neutral condition was performed after indicated 3mM MMS exposure.E, Tail moment was quantified using Comet Assay IV Lite software for n=50 cells from D.F, Comet assay under alkaline condition was performed after indicated 3mM MMS exposure.G, Tail moment was quantified using Comet Assay IV Lite software for n-=50 cells from F.H, U2OS MPG-KO cells were used for immunofluorescent microscopy using DAPI (nuclei staining), γH2AX (AF488), and 53BP1 (AF594) after exposing cells to one, two, and three hours of 3mM MMS respectively.I, Overlapping/colocalizing γH2AX-53BP1 foci were quantified on a per nuclei basis using n=50 cells from H.J, γH2AX foci per nuclei were quantified for n=50 cells from H.K, U2OS APE1-KO cells were used for immunofluorescent microscopy using DAPI (nuclei staining), γH2AX (AF488), and 53BP1 (AF594) after exposing cells to one, two, and three hours of 3mM MMS respectively.L Overlapping/colocalizing γH2AX-53BP1 foci were quantified on a per nuclei basis using n=50 cells from K.M, γH2AX foci per nuclei were quantified for n=50 cells from K.N, Immunoblot confirmation of APE1-KO cells was performed in parallel with WT U2OS cells.For all experiments, the Kolmogorov-Smirnov test was used to determine statistical significance and is demonstrated as follows: ****, p<0.0001; ns, no significance. Scale bar = 10μm.
Fig 2: NAD+ availability and PARP1-dependent PAR production promote MMS-induced DSB formation.A, PAR (magenta) was imaged using immunofluorescence (AF488) and DAPI (nuclei) staining after indicated exposure to 3mM MMS in both WT and PARP1-KO U2OS cells.B, U2OS PARP1-KO cells were treated with MMS in parallel with WT U2OS cells and stained for DAPI, 53BP1 (AF594) and γH2AX (AF488).C, Overlapping/colocalizing γH2AX-53BP1 foci were quantified on a per nuclei basis using n=50 cells from B.D, PAR was imaged after supplementation with Nicotinamide Riboside Chloride (NR) (200μM for 24 hours pre-treatment) and subsequent MMS challenge.E, NR treatment was performed before immunofluorescent microscopy using DAPI (nuclei staining), γH2AX (AF488), and 53BP1 (AF594) after exposing cells to one, two, and three hours of 3mM MMS respectively in parallel with untreated controls.F, Overlapping/colocalizing γH2AX-53BP1 foci were quantified on a per nuclei basis using n=50 cells from E.G, PAR was imaged after treatment with FK866 (50μM for 24 hours pre-treatment) and MMS challenge as indicated.H FK866 treatment was performed before immunofluorescent microscopy using DAPI (nuclei staining), γH2AX (AF488), and 53BP1 (AF594) after exposing cells to one, two, and three hours of 3mM MMS, respectively, in parallel with untreated controls.I Overlapping/colocalizing γH2AX-53BP1 foci were quantified on a per nuclei basis using n=50 cells from H.For all experiments, Kolmogorov-Smirnov test was used to determine statistical significance, and statistical significance is demonstrated as follows: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, no significance. Scale bar = 10μm.
Supplier Page from Novus Biologicals, a Bio-Techne Brand for 53BP1 Antibody [Alexa Fluor® 594]
Available conjugates: Alexa Fluor 405;DyLight 350;Alexa Fluor 532;Alexa Fluor 594