(a) Experimental design for a chemically induced skin tumorigenesis study. Mice were treated with three DMBA doses for three consecutive weeks. Tumors larger than 1 mm in diameter were counted weekly. At week 31, tumors were collected for analysis. Dlx3cKO; n=12: Control; n=10 (b) Dorsal view of control and Dlx3cKO mice after 31 weeks of DMBA treatment. (c) H&E staining of sections of skin biopsies 31 weeks after DMBA treatment shows that three doses of DMBA treatment significantly increases hyperplastic epidermis, papilloma and carcinoma in situ of Dlx3cKO, but not that of control animals (Scale bars, 1mm). (d) Number of tumors per mouse. (e) Tumor incidence: Comparison of control and Dlx3cKO incidence curves. The difference in tumor incidence between wild-type and knock-out mice becomes significant from week 17 onwards; ***P-value <0.001, Student’s t-test. (f) Frequencies of skin tumors: 3x DMBA-only treatment increased numbers of skin tumors only in Dlx3cKO mice. (g) Representative immunohistochemistry of DLX3, K13 and PCNA (Scale bars, 100μm and 10μm). As shown DLX3 expression is absent in the skin papilloma. Higher PCNA expression in nucleus and elevated K13 expression in cytoplasm were detected in papillomas from Dlx3cKO mice. (h) Detection of proliferating cell nuclear antigen (PCNA)-positive cells in tumor tissue with the control skin. Data are presented as mean ± standard error of the mean. (*** P-value <0.001). n=number of mice.