room temperature for 2 h in a dark box. The sections were then washed, stained with Hoechst 33258 (1 mg/ml) for 2 min, and viewed using a confocal microscope (FluoViewTM FV300, OLYMPUS). The following primary and secondary antibodies were used: NFATc2 (Upstate, 07-136, rabbit antiserum, 1:100), NFATc1 (Santa Cruz, 7A6, mouse, 1:100), and cyclin G2 (Abcam, ab5502, rabbit polyclonal, 1:200). Anti-mouse or anti-rabbit IgG (Molecular Probes, Alexa Fluor 488- or 555- conjugated goat IgG, 1:200) were used as secondary antibodies. For immunocytochemistry, PHK cells were cultured on glass- bottomed dishes coated with human Type-1 collagen and grown to 80% confluence. After incubation in high [Ca2+] medium for 4 h, the cells were immediately fixed with 4% PFA in PB, permeabilized with 0.5% Triton X-100 and blocked with 10% BSA. The following primary and fluorochrome-conjugated secondary antibodies were used: NFATc2 (Upstate, 07-136, rabbit antiserum, 1:250) and anti-rabbit IgG (Molecular Probes, Alexa Fluor 488-conjugated goat IgG, 1:500). Western Blotting Analysis Skins were excised from male Wistar rats at postnatal day 7 and the epidermal and dermal layers were separated with dispase at 4uC overnight. The isolated samples were frozen in liquid nitrogen, pulverized and lysed in lysis buffer, followed by boiling in sample buffer (50 mM Tris-HCl, pH 6.8, 5% glycerol, 1% SDS, 0.1% BPB, 2% 2-ME). PHK 16-0b cells and HEK293 cells were harvested at 80% confluence. After sonication, cell lysates were boiled in sample buffer. The cell and tissue lysates were separated by SDS-PAGE on a 10% acrylamide gel and transferred to a nitrocellulose membrane using the iBlot Dry Blotting System (Invitrogen). After blocking with 5% skim milk in TBST, the blots were incubated with primary antibodies against the following proteins: CnA (epitope: the middle (246–265) of the amino acid sequence, SPA-610, Stressgen Bioreagents Corp.), CnAß (epitope: the N-terminus (18–28) of the amino acid sequence, AB1697, Lot No. 0612047223, Chemicon), cyclin G2 (1572-1, Epitomics and ab5502, Abcam), p21 (sc-756, Santa Cruz), ß-actin (A-5316, Sigma), and GAPDH (sc-32233, Santa Cruz). These primary antibodies and HRP-conjugated secondary antibodies (Sigma) were appropriately diluted according to manufacturer’s recom- mendation. Microarray Analysis Microarray analysis was performed using a human 44K whole genome oligo DNA chip (Agilent Technologies) as described previously [21]. Briefly, mRNAs (150 ng) from PHK cells treated with 1 mM CsA or 3 mM 11R-VIVIT for 24 h were pooled into one master total RNA mix, and labeled with Cy-3 or Cy-5 using an Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent). Hybridization and wash processes were performed according to the manufacturer’s instructions, and hybridized microarrays were scanned using an Agilent Microarray scanner G2565BA. For detection of genes with significant differential expression between the control group and those treated with CsA or 11R-VIVIT, each slide image was processed using the Agilent Feature Extraction ver.8.5.1.1. All data is MIAME compliant and the raw data has been deposited in a MIAME compliant database, Gene Expression Omnibus (accession number GSE25175). Proliferation Assay and Vector Constructs The effect of cyclin G2 expression on keratinocyte proliferation was analyzed in PHK cells using a WST-1 assay (Roche) as described previously [22]. Recombinant adenovirus containing human ccng2 (cyclin G2) cDNA, chicken ß-actin promoter and Calcineurin Activation and Hair Development SV40 polyadenylation signal was produced using the Adenovirus Expression Vector Kit (Takara) as described previously [22]. Human ccng2 gene was purchased from ATCC (item number, 9117362; GenBank, BC032518). The LacZ recombinant adeno- virus was also produced following the manufacturer’s protocol. For the transient gene expression of CnAß-FK in HeLa cells, the open reading frame of CnAß-FK cDNA was subcloned into the pcDNA 3.1/V5-His vector (Invitrogen) using a TOPO TA Expression Kit (Invitrogen) and the cells were transfected with the plasmids using Lipofectamine 2000 (Invitrogen). Statistics Data are shown as means 6 S.E. The data were analyzed either by Student’s t test to identify significant differences between two groups or by Scheffe’s posttest analysis following two-way ANOVA to compare multiple groups. P values less than 0.05 were considered significant. Results Molecular Characteristics of a Novel Calcineurin Aß Isoform To investigate the expression of calcineurin A (CnA) in hair follicles (HF), we first examined CnA expression in primary human keratinocyte (PHK) cells. Western blot analyses using anti-CnA antibodies, which recognize the alpha and ß isoforms of CnA, identified two bands in PHK cells: an atypical CnA band (,48 kDa) and the typical CnA (,60 kDa) band (Fig. 1A). The atypical CnA band was not observed in other organs including mouse brain (Fig. 1A), liver, heart, kidney and lung (data not shown). This band could stably be detected using anti-CnAß antibodies (Fig. 1A), suggesting that it represents a ß isoform of CnA. We previously reported that calpain, a Ca2+-activated neutral cysteine protease, cleaved CnA alpha and generated a truncation product with a molecular mass of ,48 kDa in excessively excited neurons and in Alzheimer’s brain tissue [15,23]. We therefore postulated that the atypical CnAß was a truncated product of CnAß produced by calpain. To investigate this possibility, PHK cells were treated with ALLM, a calpain inhibitor. However, the expression of the 48 kDa and 60 kDa CnAb were not changed by this treatment (Fig. 1A). Moreover, Ca2+ stimulation did not alter 48 kDa CnA expression and no additional bands were observed in PHK cells (Fig. 1A). These results suggested that the atypical CnA was not generated by calpain. Next, we determined whether the atypical CnAß was expressed in dermis containing HF and in epidermis without HF in rat skin. Both the atypical and typical CnAß were observed in rat dermis whereas only typical CnAß was expressed in rat epidermis (Fig. 1B). These results suggested that the atypical CnAß might be expressed in hair follicles but not in the epidermis. To investigate the molecular characteristics of the atypical CnAß, we constructed a cDNA library from PHK cells and cloned a splicing variant of CnAß (CnAß-FK) corresponding to the 48 kDa CnAß. Sequence analysis of the CnAß-FK cDNA revealed that it was composed of CnAß coding sequence from exons 1–11 (1271 bp), intron 11 (1669 bp), exon 12 (98 bp), and part of intron 12 (930 bp) ending with a poly A tail (Fig. S1). A stop codon was present at the junction of exon 11 and intron 11 (Figure 1D & S1). As a result, the intron-retaining mRNA encoded a truncated CnAß that lacked the autoinhibitory domain (Figure 1C & S2). It was previously shown that artificial truncated form of CnA, which also lacked an autoinhibitory domain, had Ca2+-indepen- dent Cn activity [15,24]. To investigate whether CnAß-FK showed Ca2+-independent activity, we examined Cn activity in PLoS ONE | www.plosone.org 3 March 2011 | Volume 6 | Issue 3 | e17685