Figure 2. Nras^Q61R mutation-driven congenital nevi are initially proliferative and subsequently become senescent.

(A, B) Representative images of proliferative nevi. (C, D) Representative images of senescent nevi. Paraffin sections were immunostained for p16^Ink4a (A and C, red), MelanA/MART1 (A and C, green), Ki67 (B and D, red), or DCT (B and D, green). Yellow arrows indicate double-positive cells, and white arrows indicate single-positive cells. Dorsal skin samples from Dct-Cre Nras^Q61R/Q61R mutant mice were harvested at the ages indicated in E. Samples from three mice of each age group, with five to ten of non-adjacent samples per mouse, were quantified. Nevus cell proliferation and senescence were measured by immunofluorescence staining for Ki67 and p16^Ink4a, respectively. Quantification of the above staining revealed a statistically significant decrease in proliferative nevus cell between 10 and 20 days of age (E, red). The percentage of senescent nevus cells gradually increases between age groups until mice reach approximately 1 month of age (E, blue). Red and blue colored dots indicate mean percentages of nevus cells that are proliferative (left Y-axis) or senescent (right Y-axis), respectively. The bars represent the standard error of the mean (SEM). P-values were calculated using one-way ANOVA with Tukey’s multiple comparison test (****P<0.0001).