Expression of cell cycle checkpoint genes. A. Expression of p21 and p53 in Mrad1+/+ and Mrad1+/- keratinocytes. Western blotting analysis of p21 and p53 protein levels in keratinocytes incubated for four days after isolation. The first two lanes are the protein levels in cells without DMBA treatment, and the last two lanes are proteins from cells treated with 0.15 μg/ml DMBA for 24 hours. The data in the figure represent results from three independent western blotting experiments. +/+ and +/- indicate Mrad1+/+ and Mrad1+/- genotypes, respectively. B. Mrad9 and Mhus1 expression levels in Mrad1+/+ and Mrad1+/- keratinocytes. The gene expression levels were analyzed with real-time quantitative RT-PCR. Each result is the average ratio of the PCR results of an indicated gene relative to β-actin level for three independent samples, and each PCR result is the mean of triplicate PCR of the same sample. The difference of the Mrad1 expression levels between Mrad1+/- and Mrad1+/- cells is statistically significant (n = 3, P = 0.022). Either Mrad9 or Mhus1 expression levels were similar in both Mrad1+/+ and Mrad1+/- keratinocytes. C. Mrad1 expression levels in Mrad1+/+ and Mrad1+/- tumors. The expression levels of Mrad1 were analyzed by PCR as in B in this figure legend, and no statistical significance (n = 3, P = 0.34).