Fusako Sato, Gyula Soos, Charles Link, Kenzo Sato

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Cystic Fibrosis Transport Regulator and its mRNA are Expressed in Human Epidermis Fusako Sato, Gyula Soos, Charles Link, Kenzo Sato Journal of Investigative Dermatology Volume 119, Issue 6, Pages 1224-1230 (December 2002) DOI: 10.1046/j.1523-1747.2002.19601.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Schematic illustration of CFTR and CFTR mRNA and the locations of in situhybridization probes, primers for primary and nested PCR, and the epitopes for MoAbs used. N, N-terminus; C, C-terminus; TMD, transmembrane domain; NB, nucleotide binding domain; R, regulatory domain; ΔF508, the most frequent CF mutation resulting in deletion of phenylalanine at amino acid 508; single asterisk, location of epitope for MoAb for the R domain (MoAb-GR); double asterisk, MoAbs for C-terminus (MoAb-GC and MoAb-WC); hyb, hybridization. Arrows indicate the exons encoding different domains of the CFTR protein. Journal of Investigative Dermatology 2002 119, 1224-1230DOI: (10.1046/j.1523-1747.2002.19601.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Immunohistochemical staining of the CFTR R domain (using MoAb-GR) in human epidermis (A) and eccrine sweat gland (B). In this and subsequent figures: epiderm, epidermis; derm, dermis; S, secretory coil; D, duct; asterisk, upper stratum corneum. Large arrow in (A), granular layer cells and the deeper corneocytes; small arrow in (B), CFTR staining in sweat duct luminal membrane. Journal of Investigative Dermatology 2002 119, 1224-1230DOI: (10.1046/j.1523-1747.2002.19601.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Immunohistochemical staining of CFTR C-terminus using MoAb-GC in the epidermis (A) and the sweat gland (B). (C) Complete inhibition of immunostaining in the presence of the C-terminus blocking peptide. Asterisk in (A), stratum corneum; small arrows in (B), dark cells. Note staining of CFTR in the perinuclear cytoplasm of keratinocytes throughout the epidermis (A). Also note darker staining of the dark cells (small arrow in B). Journal of Investigative Dermatology 2002 119, 1224-1230DOI: (10.1046/j.1523-1747.2002.19601.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Immunohistochemical staining of CFTR C-terminus using MoAb-WC in the epidermis (A) and the sweat gland (B). (C) Complete inhibition of immunostaining in the presence of the blocking C-terminus peptide. Note the clear representation of perinuclear localization of immunostaining in the epidermal cells in (A) and predominant localization of immunostaining in the dark cells as in the previous study (Sato and Sato, 2000). Asterisk in (A), positive CFTR immunoreactivity also in the stratum corneum; large empty arrows in (B), lack of staining in myoepithelium; small arrows, rich staining in the dark cells. Journal of Investigative Dermatology 2002 119, 1224-1230DOI: (10.1046/j.1523-1747.2002.19601.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 RT-PCR of CFTR mRNA in isolated human epidermis, isolated human sweat glands, and T-84 cells, the last two tissues being used as controls. The amount of starting RNA before the first PCR was approximately normalized using 18 S RNA as a reference in each test tube. Lad, molecular weight ladder markers. Note that the epidermis showed clearly visible PCR products in both the first stage (or primary) and nested PCR as in T-84 cells. As in the previous study (Sato and Sato, 2000), nested PCR constantly yielded the 354 bp product whereas the primary PCR caused variable results (n=4) depending on the number of isolated glands available. The nested PCR products were sequenced to confirm their identity with CFTR. Journal of Investigative Dermatology 2002 119, 1224-1230DOI: (10.1046/j.1523-1747.2002.19601.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 In situ localization of CFTR mRNA in cryosections of human epidermis and the sweat gland. (A) The epidermis; (B) the sweat gland. (A1), (B1) Hybridized with the antisense RNA probe; (A2), (B2) hybridized with the sense probe (negative controls). In (A1), the small arrow indicates the absence of mRNA in corneocytes in the stratum corneum. Also note the presence of CFTR mRNA in the cytoplasm of epidermal cells throughout the living layers of the epidermis (A1) and the lack of staining in the control treated with the sense probe (A2). (B) Large empty arrow, the absence of mRNA in the luminal cytoplasm of luminal duct cells (B1); small arrows, darker staining of dark cells than in the neighboring clear cells (B1); asterisk in (B1), the absence of staining in the myoepithelial cells. The bars are 20 μm for all figures. Journal of Investigative Dermatology 2002 119, 1224-1230DOI: (10.1046/j.1523-1747.2002.19601.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions