Fig. S10

Detection of DNA damage response and DNA synthesis in spns1 mutants in the presence or absence of p53. (A) γH2AX- and BrdU detection in spns1 mutants in p53- and DNA damage-dependent manners. As shown in the green fluorescent panels, unaltered γH2AX intensities between spns1^+/+ and spns1^hi891/hi891 (spns1^-/-) were apparent irrespective of p53 status without UV irradiation. Increased γH2AX intensities in response to UV irradiation were observed in the presence of p53 regardless of Spns1 status. Of note, certain basal increases of γH2AX intensities were detected in the p53 mutant background. As shown in the red fluorescent panels, reduced BrdU incorporation in spns1^hi891/hi891 animals was detected in either normal or mutant p53 condition in the absence of UV treatment. UV-induced inhibition of DNA synthesis (reduction of BrdU signals) is apparently seen only in the normal p53 situation. The UV (18 mj/cm²) treatment was done at 68 hpf, followed by the phenotype observations at 72 hpf. Scale bar in the large image, 250 μm. Scale bar in the small merged image and inset, 10 μm. (B) Quantification of the γH2AX fluorescence intensities shown in (A). Quantification of data presented in panel A (n = 12) is shown in the right graph; the number (n) of animals is for each genotype. Three independent areas (periderm or basal epidermal cells in the trunk) were selected from individual animals. (C) Quantification of the BrdU-positive cells [in 25.6±2.2×10⁴ μm areas; the trunk region starting from the rostral start point of the yolk extension (the distal end of the yolk) through the end of the caudal fin] shown in (A). Error bars represent the mean ± S.D., **p<0.005; *p<0.05; ns, not significant.