Fig. 2: Loss of CPDphr function in medaka results in increased DNA damage and cell mortality upon oxidative stress.

Immunofluorescence assays measuring γ-H2AX levels in medaka wild type (grey truncated violin (WT)) as well as CPD (blue truncated violin (a)), DASH (green truncated violin (b)) and 6-4 (red truncated violin (c)) photolyase mutant (KO) cells treated with various concentrations of H₂O₂ as indicated on the x-axes. d Immunofluorescence assay of γ-H2AX levels in wild-type medaka (iCab) and photolyase mutant (KO) fish fin clips treated with 1000 µM H₂O₂ or untreated controls (Ctrl) as indicated on the x-axis. Violin plots represent individual data points (a–c, n = 6 biologically independent samples; d, n = 9 biologically independent samples) shown as black hollow circles and their probable distribution. The horizontal black lines within the violin plots represent mean values of γ-H2AX fluorescence intensity. e Quantification of automated high-throughput microscopy (AHM) assay results in medaka CPD WT and KO cells exposed to a range of H₂O₂ concentrations up to 3000 µM. Total numbers of dead and viable cells are plotted as means ± SEM (n = 4 biologically independent samples), normalized in relation to untreated cells (Ctrl) on the y-axis, while H₂O₂ concentrations are indicated on the x-axis. Note that the overall reduced cell counts in the right panel (CPD KO) compared with the left panel (CPD WT) may be a consequence of reduced cell proliferation and higher levels of cell death in the mutant cells resulting in their detachment from the cell culture surface. f Cell viability assay of medaka CPD WT and KO cells exposed to a range of H₂O₂ concentrations from 200 to 3000 µM. Mean percentage ± SEM (n = 8 biologically independent samples) of cell viability with respect to untreated cells are plotted on the y-axis, while H₂O₂ concentrations are indicated on the x-axis. All assays were performed in constant darkness (DD) and repeated at least 3 times, independently. For the cell viability assays (e, f), representative data is shown. The statistical test used for (a–d) is student’s t-test, while the test for (e, f) is two-way analysis of variance (ANOVA) test. Statistical differences (P values) are indicated in each panel. Source data are provided as a Source Data file.