Extended Data Fig. 6: The AraC-induced increase in γ-H2AX in Purkinje cells is TET- and TDG-dependent.
a, Representative examples of imaging flow cytometric analysis of Purkinje cell nuclei. BF (bright field), NeuN (green), γ-H2AX (yellow), ITPR-1 (red), DAPI (blue) and Merge (γ-H2AX and ITPR-1). TDG knockout (Tdg−/−) was induced by intraperitoneal injection of tamoxifen for CreERT2 TDGfl/fl mice. Pcp2-Cre Tet1/2/3fl/fl mice were used for Purkinje-specific knockout of Tet1/2/3 (Tet−/−). b, Quantification of γ-H2AX foci from imaging flow cytometric analysis. The genotype and treatment are indicated on the bottom. Data are presented as median with interquartile range, n = 110 and 579 (WT), 256 and 898 (WT with AraC), 101 and 129 (Tdg−/− with AraC), and 352 and 314 (Tet−/− with AraC) nuclei examined in 2 independent experiments. Statistical significance was determined using two-sided Mann-Whitney test, p value is indicated (p < 1e-15). c, Flow cytometric analysis of nuclei isolated from 6-month old mouse cerebellum after PBS or AraC treatment. Y- and X-axes indicate the expression of the Purkinje specific marker ITPR-1 and the DNA damage marker γ-H2AX respectively. γ-H2AX positive Purkinje cells are marked by red. For the flow cytometry gating strategy, see Supplementary Fig. 2. d,e, Quantification of γ-H2AX level by flow cytometry. The genotypes and treatments are indicated. Pcp2-Cre Tet1/2/3fl/fl mice were used for Purkinje-specific knockout of Tet1/2/3 (Tet−/−) in (e). For the flow cytometry gating strategy, see Supplementary Fig. 2.
