Zmpste24 deficiency disrupts signaling pathways implicated in stem cell regulation in the hair follicle. (A) Immunoblot analysis of levels of β-catenin, active β-catenin, and Mitf in the epidermis of the indicated mice. β-Actin was analyzed as a loading control. Results are representative of three different animals per genotype. (B and C) Distribution of β-catenin and Act–β-catenin in tail skin hair follicles from Zmpste24^+/+ and Zmpste24^−/− mice. (B) Boxed areas are magnified in the bottom panels. (D) Quantification of cyclin D1 and Hoxc13mRNA by quantitative RT-PCR. Data were normalized to actin expression. (E) RNAi experiments in Pam212 cells using Zmpste24-specific or scrambled siRNA. (left) Immunoblot analysis showing Zmpste24 protein reduction after 72 h of siRNA treatment. Results are representative of three experiments. (middle and right) Normalized luciferase/renilla activities of reporter vectors transiently transfected in Pam212 cells containing either multimerized promoter sequences recognized by β-catenin–lef/tcf complexes (pTOPFLASH; TOP), the same mutated sequences (pFOPFLASH; FOP), or the human cyclin D1 promoter. Assays were performed in triplicate. (F) Distribution of Mitf in the skin from Zmpste24^+/+ and Zmpste24^−/− mice. (G) Quantification of mRNA expression by quantitative RT-PCR of dopachrome tautomerase and Tyrp1. **, P < 0.01. Error bars represent SEM. Bars: (B) 100 μm; (C) 20 μm; (F) 50 μm.