Calcineurin Activation and Hair Development Figure 4. Effects of 11R-VIVIT on hair growth induction in nude mice. (A) The vertex region (blue circle) was chosen as the treatment spot with 11R-VIVIT. (B) Macroscopic and microscopic assessment of hair growth induction. Upper panels, control mouse treated with ointment only. Lower panels, 11R-VIVIT-treated mouse. Right panels, longitudinal sections stained with H. E. Scale bar, 1 mm. (C) Histological features of the skin of control and 11R-VIVIT-treated mice. i–iv, control mice; v–viii, 11R-VIVIT-treated mice. Longitudinal (i, ii, v and vi) and transverse (iii, iv, vii and viii) sections stained with H.E. (i, iii, v and vii), and the same sections assessed for birefringent hair shafts using a polarizing microscope (ii, iv, vi and viii). Scale bars in ii and v, 500 mm; in iv and vii, 200 mm. (D) Hair growth in control (2) and 11R-VIVIT-treated (+) mice was quantified using a polarizing microscope by counting the number of hair follicles containing birefringent hair shafts. n = 10 each. *P,0.0005. doi:10.1371/journal.pone.0017685.g004 (Fig. 5E). We also screened skin from Wistar rats that had been treated with CsA for three successive days from postnatal day 24 (anagen stage) for the subcellular localization of NFATc2 and cyclin G2 expression (Fig. S8). In vehicle-treated rats, NFATc2 was observed in both nuclei (arrows in Fig. S8C) and cytoplasm of follicular keratinocytes in the HF. Cyclin G2 expression was also detected in many of the follicular keratinocytes in the HF of control rats (Fig. S8E). In contrast, NFATc2 was mainly detected in the cytoplasm of follicular keratinocytes of CsA-treated rats (Fig. S8F and G). A low level of cyclin G2 expression was observed in the follicular keratinocytes of CsA-treated rats (Fig. S8I). Discussion The physiological functions of the CN/NFAT signaling pathway have been investigated in diverse organs including the immune system, the neuronal system and the hair follicle. In the hair follicle, the pathway is functionally active and its inhibition enhances hair growth [4,5]. A recent study elucidated many details of the molecular mechanism of this pathway in hair stem cells [5]. It was found that NFATc1 was specifically expressed and that the Cn/NFATc1 pathway down-regulated proliferation of hair stem cells [5]. However, the precise molecular mechanisms involved in PLoS ONE | www.plosone.org 8 March 2011 | Volume 6 | Issue 3 | e17685