Figure 6. Immune and melanocyte proliferative responses post UVR in different mouse strains.

(A) Parameters measured post UVR in different mouse strains. Top bar graph shows number of cells (stained with anti-CPD antibody) positive for pyrimidine dimers at 1 (D1), 4 (D4) and 7 (D7) in whole skin after neonatal UVR. Second bar graph shows the number of epidermal melanocytes (stained with anti-Sox10 antibody) per field. Third graph shows the number of dermal neutrophils (stained with anti-myeloperoxidase) per field. Fourth graph shows the number of dermal macrophages (stained with anti-F4/80 antibody) per field. For each replicate time-point cells were counted from at least three mice, with at least 10 fields counted per mouse. (B) Levels of each measured parameter post UVR presented as a heatmap. Strains are separated according to whether they carry the Rrp15 susceptibility allele (NOD, AJ, FVB) or not (DBA, 129S, (B6). For each parameter, groups were treated separately and analysed for significant differences between groups using the Mann-Whitney U test using PRISM. (C) Heatmap using the same data as in the previous heatmap, but this time strains are listed according to age of onset of melanoma (fastest to slowest), and each parameter analysed for Pearson correlation co-efficient with age of onset of melanoma using PRISM. P-value for a two-tailed test based on 95% confidence interval. Final row shows correlation Pearson r value and p value for the correlation. (D) Both panels show myeloperoxidase (MPO) staining of neonatal FVB mouse skin at 24 hr post UVR. Yellow arrows denote neutrophils, which were present in PBS-treated skin but not skin treated with neutrophil depleting Ly6g antibody. (E) Graph shows average number of neutrophils per field in UVR treated skin with (n = 17) or without (n = 15) treatment with neutrophil depleting antibody. p-value calculated using the Mann Whitney U test. Mice were collected from over a number of litters, with each litter divided in two to randomly establish treatment and control groups. (F) Kaplan-Meier analysis of melanoma free survival in neonatal UVR-treated mice. We aimed to study at least 20 mice in each group. 20 animals per group is sufficient to detect a difference in penetrance of 40% with statistical power of 80%. Actual numbers analysed n = 18 for the neutrophil-depleted group, and n = 15 for the control group. Significance of differences between groups calculated using the Log rank (Mantel Cox) test. There was no significant difference in melanoma age of onset whether or not neonates were treated with neutrophil depleting Ly6g antibody.