(A and B) Immunostaining for ssDNA, PAR, and CD206 (A) and the quantification of ssDNA-, PAR-, CD206-positive microglial cells (B) in Mutyh^+/+ (n = 6, each) or Mutyh^−/− (n = 6, each) microglia. (A) ssDNA (green) and DAPI (blue) staining (upper panel), PAR (green) and DAPI (blue) staining (middle panel), and CD206 (green) and DAPI (blue) staining (lower panel). (B) Quantification of the ratio of ssDNA-positive microglial cells (upper graph), PAR-positive microglial cells (middle graph) and CD206-positive microglial cells (lower graph). Microglial cells were treated in the presence or absence of 250 μM menadione for 24 hr. More than 100 microglial cells per well were evaluated. Mutyh deficiency suppressed the SSBs and PAR accumulation and prevented the reduction of CD206 expression after menadione exposure. Scale bar: 20 μm. (C) ELISA for TNF-α in the supernatants of Mutyh^+/+ (n = 7) or Mutyh^−/− (n = 6) microglia. Microglial cells were treated with menadione for 24 hr at the indicated concentrations. (D) Cell toxicity of microglial cells to 661W photoreceptor cells. Mutyh^+/+ (n = 8) or Mutyh^−/− (n = 14) microglia were treated with menadione for 6 hr at the indicated concentrations. After being washed with PBS, the activated microglia were cocultured with 661W photoreceptor cells for 24 hr and the supernatants were subjected to an LDH assay. Mutyh deficiency reduced the cell toxicity of microglial cells induced by menadione. Figures shown are the representative results from 3 experiments (A), and the data shown are the combined results of 3–4 experiments (B–D). Data are presented as whisker-box plots. The central horizontal bars indicate the medians, boxes indicate 25th to 75th percentiles, and whiskers indicate 1.5 times the interquartile range from the bottom and the top of the box (B and C). Outliers are shown as dots. Error bar: mean ±SEM (D). Student’s t tests were performed to assess the significance. *P < 0.05, **P < 0.01.