Fig. 9: Excision of oxidized bases induces SSB to DSB conversion in vitro.

Three experimental tests for SSB to DSB conversion. More than n = 50 NIH3T3 were identified as neurons by NeuN staining and positive cells were subsequently imaged for γH2AX or for 53BP1 in each treatment regime for n = 3 replica platings. Scale bars were 10 µm for all images. a Schematic design for experiment 1 as described in text. NIH3T3 cells were transfected with “sense” major satellite guide RNA (sgMajSat) and treated with peroxide for 30 min with (+XJB-5-131, 200 µm) or without XJB-5-131 (−XJB-5-131). The sgMajSat* transfection was included in experiment 1 only to ensure that DSB quantification in (b, e, h) occurred under the same conditions. b (Top panel −XJB-5-131); IF results for DSB formation in selected cells as detected by staining intensity with the γH2AX (green, middle panel) and 53BP1 (red, right panel) DSB markers antibodies. An overlay of the two (yellow foci, left panel) in the absence of XJB-5-131. (Bottom, +XJB-5-131). Same as (top) but in the presence of XJB-5-131. The low blue emission from mBFP expressed from the sgMajSat* RNA expression vector is represented as gray so as not to be confused with DAPI (blue). c Quantification of (b) in n = 50 cells; gray (−XJB-5-131) and black (−XJB-5-131) in n = 3 replicate platings. d Schematic design for experiment 2 as described in text. XJB-5-131 inhibition of DSBs requires base oxidation. e IF result for DSBs after transfection of the CASD10A nickase targeted to major satellite DNA using sgMajSat RNA guide ± XJB-5-131, as indicated. The IF signals of γH2A-X (green, middle panel) and 53BP1 (red, right panel) target and co-localized at major satellite loci in heterochromatin domains⁸⁸ (see j) within 6 h post transfection ± XJB-5-131. f Same as (c) but for Quantification of n = 50 cells from (e). g Schematic design for experiment 3 as described in text. h Same as (c) but for CAS9. i Quantification of 50 cells from (i) is as described in (f). Data are expressed as median (line), the box indicating the 25th and 75th percentile with whiskers indicating the minimum and maximum values. The probability statistics in (c), (f), and (I) were derived from a one-way ANOVA, ****P < 0.0001 for all cell type comparisons in (c, f, i). j Schematic image of heterochromatin domains. k IF detection of heterochromatin domains detected by the histone H3K9Me3 antibody (right panel), DAPI (middle panel), and an overlay of both (left panel). NIH3T3 cells have heterochromatin domains and stained positively with the histone H3K9Me3 antibody. Shown is a representative example. This was checked in >10 cells from each of 3 separate cell cultures, Scale bars represent 10 µm.