Figure Legends
Figure 1. KY19382 increases hair induction activity via activation of Wnt/β-catenin pathway. (a) TOPflash activity of HEK293T cells treated with the indicated concentrations of KY19382, I3O or DMSO for 24 hours (n=6). (b-e) Human DP cells were treated with the indicated concentrations of KY19382, 100 μM MNX, or DMSO for 48 hours. (b) Cell viability of human DP cells (n=4). (c) Immunoblotting analysis for β-catenin, α-tubulin, p-GSK3β and PCNA were analyzed using human DP cells. (d) Immunocytochemical staining for β-catenin. Nuclei were counterstained with DAPI (blue). Arrows indicate nuclei. (n=12) (e) ALP staining and (f) ALP activity test (n=3). (g) Transfected cells were subjected to ALP staining. (h) ALP activity was quantified (n=6). Scale bars = 100 µm. Values are expressed as the means±SEM. Student’s t-test (* P < 0.05, ** P < 0.01, *** P < 0.001).
Figure 2. KY19382 accelerates mouse vibrissa follicle elongation. Mouse vibrissa follicles were cultured with 5 μM KY19382 or 100 μM MNX for 6 days. (a) The length of vibrissa follicles was measured at 6 days after treatment with 5 μM KY19382 or 100 μM MNX. Elongation rate of mouse vibrissa follicles was calculated as the difference in the length of vibrissa follicles wherein the vibrissa follicle length in the control group at day 6 was considered 100%. Scale bars = 200 µm. (b) IHC analysis of mouse vibrissa follicle hair bulb for β-catenin and Ki67. Dashed lines mean mouse vibrissa follicles. (c) Mouse vibrissa follicles were subjected to ALP staining. Arrow indicates ALP-positive region. (d) X-gal staining of vibrissa follicles fromAxin2lacZ/+ mice. Arrow indicates X-gal-positive region. (b-d) Scale bars = 100 µm. Student’s t-test (* P < 0.05, ** P < 0.01, *** P < 0.001, n=5).
Figure 3. KY19382 stimulates hair regrowth in vivo. Mice were treated with 0.5 mM KY19382 or 100 mM MNX for 14 or 28 days. (a) Gross image showing hair re-growth in mice treated with each drug for 28 d and quantitative measurements of weight of regenerated hairs. (b) H&E staining to evaluate the hair follicles of skins with different treatments and relative number of hair follicles and dermal thickness. (c) IHC staining for keratin 15, β-catenin, and Ki67 using the dorsal skin of mice treated with each drug for 14 days. Lines show keratin 15-positive bulge stem cells region. (d) Immunoblotting analysis for β-catenin, PCNA, and ERK. (e) ALP staining was performed to evaluate the ALP activity of hair follicles with different treatments. Arrow points to ALP-positive region. Scale bars = 100 µm. Values are expressed as the means±SEM. Student’s t-test (* P < 0.05, ** P < 0.01, *** P < 0.001, n=5).
Figure 4. KY19382 stimulates WIHN in vivo. Mice wounds were treated with 0.5 mM KY19382 or 100 mM MNX for 14, 25, or 40 days. (a) ALP staining to evaluate the neogenic follicles of mice treated with each drug for 25 days and quantitative measurements of ALP-positive neogenic follicles. Dashed lines show ALP-positive neogenic hair follicles. (b) H&E staining to evaluate newly formed hair follicles in mice treated with each drug for 14 days and the relative number of newly formed hair follicles. Dashed lines mean boundary between epidermis and dermis. Arrows show newly formed follicles (c) IHC staining for fgf 9, keratin 17, β-catenin, Ki67, and PCNA. (d) Immunoblotting analysis for fgf 9, keratin 17, β-catenin, PCNA, and ERK. (e) Gross images show newly formed hair in mice after treatment for 40 days. Scale bars = 100 µm. Values are expressed as means±SEM. Student’s t-tests (* P < 0.05, ** P < 0.01, *** P < 0.001, n=6).
Figure 5. KY19382 induces hair follicle neogenesis in hairless mice. Mouse dermal cells were pretreated with 5 μM KY19382 for 72 hours and then injected into hairless mice. The hair follicle neogenesis was analyzed on at 14 days after transplantation. (a) Hair follicle neogenesis at injected area of hairless mice. Arrow indicates hair regeneration area on the skin. (b) Magnified image of area exhibiting hair follicle neogenesis and quantitative analysis of regenerated hair follicles. (c) IHC analysis for β-catenin (scale bars = 50 µm) and quantitative analysis. Dashed lines mean hair follicles. Values are expressed as means±SEM. Student’s t-tests (* P < 0.05, ** P < 0.01, *** P < 0.001).