Dihydrotestosterone-Inducible Dickkopf 1 from Balding Dermal Papilla Cells Causes Apoptosis in Follicular Keratinocytes Mi Hee Kwack, Young Kwan Sung,

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Dihydrotestosterone-Inducible Dickkopf 1 from Balding Dermal Papilla Cells Causes Apoptosis in Follicular Keratinocytes Mi Hee Kwack, Young Kwan Sung, Eun Jung Chung, Sang Uk Im, Ji Seop Ahn, Moon Kyu Kim, Jung Chul Kim Journal of Investigative Dermatology Volume 128, Issue 2, Pages 262-269 (February 2008) DOI: 10.1038/sj.jid.5700999 Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Expression of AR in cultured balding DP cells. Cells were immunostained in the (a) absence or (c, e, g) presence of AR antibody. (a, c) Cells from passage 2, (e) passage 4, and (g) passage 8 are shown here. (b, d, f, h) Corresponding 4,6-diamidino-2-phenylindole nuclear staining is also shown. (i) RT-PCR analysis also shows that AR mRNA level is gradually decreased during the subcultivation. Bar=0.1mm. Journal of Investigative Dermatology 2008 128, 262-269DOI: (10.1038/sj.jid.5700999) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 DKK-1 induction in response to DHT in balding DP cells. (a) Cells were treated with varying concentrations of DHT for 6hours and analyzed by RT-PCR and (b) relative level of DKK-1 is shown as mean±SD of three experiments. (c) Cells were also treated with 100nM DHT for varying times and (d) mean±SD of three experiments is shown. The concentrations of DKK-1 in conditioned medium were measured by ELISA. Cells were treated with varying concentrations of DHT for 4 days, and (e) data are expressed as means±SD of three experiments. Journal of Investigative Dermatology 2008 128, 262-269DOI: (10.1038/sj.jid.5700999) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Effect of DHT on the growth of ORS cells co-cultured with DP cells. (a) ORS cells were co-cultured in the presence or absence of DHT for 4 days and viable ORS cells were counted. Data are expressed as means±SD of three determinations per experiment from three independent experiments (*P<0.05). (b) A DNA histogram shows DNA content of co-cultured ORS cells in the absence (left) or presence (right) of 100nM DHT. Journal of Investigative Dermatology 2008 128, 262-269DOI: (10.1038/sj.jid.5700999) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Effect of neutralizing antibody against DKK-1 on the DHT-induced growth inhibition of ORS cells. DP and ORS cells were co-cultured in the absence (lane 1) or presence (lane 2–4) of DHT for 4 days with 200ng/ml preimmune goat IgG (lane 3) or 200ng/ml anti-DKK1 antibody (lane 4). Data are expressed as means±SD of three determinations per experiment from three experiments (*P<0.05 compared with lane 2). Journal of Investigative Dermatology 2008 128, 262-269DOI: (10.1038/sj.jid.5700999) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Effect of rhDKK-1 on the growth and apoptosis of ORS cells. Cells were treated in the absence (a) or presence of 10ng/ml (c) or 25ng/ml (e) rhDKK-1 for 24hours and stained with the TUNEL method. (b, d, f) The corresponding 4,6-diamidino-2-phenylindole nuclear staining is also shown. (g) TUNEL-positive apoptotic cells (green) were counted and data are means±SD from two independent experiments. (h) Cells were also treated with rhDKK-1 for 4 days and 3-[4,5]dimethylthiazol-2,5-diphenyltetrazolium bromide assay was employed to measure the cell viability. Data are means±SD of six determinations per experiment from three experiments (*P<0.05). Bar=0.1mm. Journal of Investigative Dermatology 2008 128, 262-269DOI: (10.1038/sj.jid.5700999) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Effect of anti-DKK1 antibody on DHT-induced cell death in cultured hair follicles. Hair follicles were cultured in (a) the absence or (b, c) the presence of 100nM DHT for 2 days and cryostat sections (7μm) were stained with hematoxylin and eosin H&E. (c) DHT-induced epithelial cell death was reversed in the presence of the 500ng/ml anti-DKK1 antibody. High-powered images of corresponding regions are also shown in (d, e, and f). Bar=0.1mm. Journal of Investigative Dermatology 2008 128, 262-269DOI: (10.1038/sj.jid.5700999) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Immunoblot of DKK-1 from balding and non-balding haired scalp of patients with AGA. (a) Matched balding and non-balding scalp lysates from four patients were pooled and probed with the goat polyclonal antibody against human DKK-1. (b) Blots were also reprobed with a biotin-conjugated goat antihuman DKK-1 antibody. (c) The membranes were also probed with antibody against actin. Journal of Investigative Dermatology 2008 128, 262-269DOI: (10.1038/sj.jid.5700999) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions