(A) CnA expression in PHK cells and mouse brain. PHK cells were treated with 1 µM ALLM or incubated with 1 mM Ca²⁺ for 24 h. (B) Expression of CnAß in the dermis and epidermis of rat. An atypical CnAß with a molecular mass of 48 kDa was observed in the dermis and PHK cells. ß -actin expression (Actin) was used as the control. (C) Comparison of the primary structure of CnAß-FK with other splicing variants of CnAß. CnAß-FK lacks an autoinhibitory domain. The gray box in variant 2 represents amino acid sequences translated from mRNAs of the intron. (D) Comparison of the nucleotide sequence of the 3′ terminus of CnAß-FK cDNA with that of variant 1. Blue box, cDNA derived from exons; red box, cDNA derived from introns. *, stop codon. (E) Ectopic expression of CnAß-FK in HEK293 cells. PHK cells lysate was loaded as a positive control. (F) Relative CnAß activity in brain lysate and each cell line. HEK 293 cells were transfected with pcDNA3.1 plasmid containing CnAß-FK cDNA and harvested 24 h later. Cn phosphatase activity was assayed in the presence or absence of 1 mM Ca²⁺ or 1 µM cyclosporin A (CsA). Phosphatase activity of each sample was standardized with that in the presence of Ca²⁺ but not CsA. Purified CnA from bovine brain was used as a positive control. n = 5 each sample. *P<0.01. (G) Subcellular distribution of NFATc2 in PHK cells and HEK293 cells. Cells were transfected with a GFP-NFATc2 plasmid and incubated for 24 h. PHK cells were then treated with 11R-VIVIT (1 mM) for 6 h or stimulated with Ca²⁺ (1 mM) for 15 min. Note that GFP signals were seen not only in the cytoplasm but also in the nucleus of control PHK cells. In HEK293 cells, under basal conditions, GFP signals were seen only in the cytoplasm. Scale bar, 10 µm.

(A) Time-scale of the hair cycle in male Wister rats. Each phase was determined using criteria described previously [42]. The intensity of the gray shading indicates the rate of proliferation of follicular keratinocytes: white<proliferation activity<black. (B–F) Distribution of CnAß-FK mRNA. B and C, telogen stage. D, late phase of anagen stage. E and F, transition stage from catagen to telogen. (C and F) Sections were counter-stained using hematoxylin (blue staining). (G–J) Results of immunohistochemical analysis of NFATc2 (green) and NFATc1 (red) in hair follicles. Cell nuclei were visualized by Hoechst staining (blue in G–I and K). (G) NFATc2 expression in hair follicle at postnatal day 28. (H) NFATc2 and NFATc1 expression in bulge and SG cells at postnatal day 28. Arrows, NFATc1 expression in bulge cells. Arrowheads, nuclear localization of NFATc2 in SG cells. (I) NFATc2 expression at postnatal day 49. Arrowheads indicate localization of NFATc2 in nuclei of SG cells. (J and K) Higher magnification image of the boxed region in G. (J) NFATc2 expression. (K) Hoechst staining. (L) Hematoxylin-eosin (H.E.) staining of hair follicles at postnatal day 28. After immunofluorescence staining, H.E. staining was performed in the same section to reveal the histology of hair follicle. DP, dermal papillae; IRS, inner root sheath; ORS, outer root sheath; SG, sebaceous gland. Scale bars in B–F, 100 µm; in B, C, and G–K, 50 µm.

(A) The microarray analysis identified 24 genes that showed down-regulated expression in PHK cells treated with either 11R-VIVIT or CsA; ccng2 (cyclin G2) expression showed the greatest decrease after Cn/NFAT inhibition. (B) Time-dependent changes in cyclin G2 and p21^waf/cip1 expression in PHK cells treated with 11R-VIVIT. (C) Quantitative analysis of the changes in expression changes of cyclin G2 and p21^waf/cip1 in PHK cells after 11R-VIVIT treatment. n = 5 for each sample group. * P<0.01, ^† P<0.05. (D) Effect of cyclin G2 overexpression on the proliferation of PHK cells. Cells were infected with recombinant adenoviruses carrying cyclin G2 and lacZ at an MOI of 100. Overexpression of cyclin G2 significantly inhibited cell proliferation compared with LacZ-infected cells (*P<0.01). (E) Expression of cyclin G2 in rat hair follicles at postnatal days 9 (first anagen stage) and 49 (second telogen stage). Arrowheads, differentiated keratinocytes in a distal part of the hair shaft; arrows, proliferating keratinocytes in the proximal hair shaft. DP, dermal papillae; HG, hair germ; SG, sebaceous gland. Scale Bar = 50 µm.

(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 µm.

(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 µm; in iv and vii, 200 µm. (D) Hair growth in control (−) 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.