Calcineurin Activation and Hair Development Figure 2. Localization of CnAß -FK mRNA in rat skin. (A) Schema of sense and anti-sense probes used for in situ hybridization (ISH). The probes correspond to the full sequence of intron 11 and are specific for CnAß-FK mRNA. Sense probes were used as the negative control. (B and C) Expression of CnAß-FK mRNA in rat skin. Skin sections from the parietal region were prepared from male Wistar rat (postnatal day 28, corresponding to the late phase of the second anagen). (D) Higher magnification micrograph to show the mRNA expression in epidermais and SG. Epi, epidermis; SG, sebaceous gland. Scale bars in B and C, 1 mm; in D, 50 mm. doi:10.1371/journal.pone.0017685.g002 expression of cell cycle-associated proteins and apoptosis-associated proteins [4]. To investigate the mechanism of regulation of the hair cycle after activation of CnAß-FK/NFATc2, we carried out a DNA microarray analysis to identify genes down-regulated by 11R-VIVIT and CsA in PHK cells. Twenty-four genes showed repressed expression after 11R-VIVIT and CsA treatments (Fig. 5A). Of these genes, the largest effect was shown by ccng2 (Fig. 5A), which encodes the unconventional cyclin G2. Cyclin G2 is an atypical cyclin that associates with protein phosphatase 2A but not proteins of the cell cycle-associated Cdk family; it is involved in cell cycle arrest and apoptosis [18,19,27]. The effect of 11R-VIVIT on time-dependent changes in cyclin G2 expression was examined in PHK cells. Cyclin G2 expression decreased with time following 11R-VIVIT application in a similar fashion to that of p21waf/cip1, which has been identified as a target protein of Cn/NFAT in the hair follicle (Fig. 5B and C). We further examined whether overexpression of CnAß-FK induced endogenous cyclin G2 in Hela cells. Exogenous CnAß-FK induced the cyclin G2 expression (Fig. S7). Moreover, overexpression of cyclin G2 inhibited the proliferation of PHK cells compared to control and LacZ-overexpressing cells (Figure 5D). Cyclin G2 was strongly expressed in the mature keratinocytes localized distally in the hair shaft (arrowheads), whereas the PLoS ONE | www.plosone.org 6 March 2011 | Volume 6 | Issue 3 | e17685