Fig 1.
Immunofluorescence analysis of CD44 expression in frozen and FFPE normal human skin.
(A) CD44s-specific mAb stained both epidermis and dermis of frozen human skin. (B, C) Only epidermis of frozen human skin was stained by CD44v6-specific mAb (B) and CD44v9-specific mAb (C). (D) Normal mouse IgG (negative control) showed no signal in either epidermis or dermis frozen human skin. (E-J) Analysis of CD44s and CD44v6 expression in FFPE samples from different anatomical locations, gender, age and ethnicity. Nuclei were counterstained with DAPI. Scale bars, 50 μm.
Table 1.
Summary of analysis of CD44s, CD44v6 and CD44v9 expression in frozen and FFPE normal human skin.
Fig 2.
PCR analysis of CD44 transcripts in human epidermis.
(A) PCR strategy. Primers were designed to amplify all variants of full-length CD44 transcripts in human epidermis. Filled boxes represent standard exons and empty boxes represent alternatively spliced exons. The non-coding exon 18 is not shown. (B, C) Analysis of PCR products of CD44 transcripts on 1% agarose gel (B) and 5–20% acrylamide gel (C).
Fig 3.
Cloning of CD44 transcripts in human epidermis.
(A) Successful cloning of different CD44 transcripts from human epidermis by PCR. (B) Summary of CD44 transcripts in human epidermis; Epidermis contains at least 10 major classes of CD44 transcripts. The numbers on the right indicate the number of variants in each class.
Fig 4.
Analysis of CD44 transcripts in human epidermis.
(A) Presentation of CD44 cDNA structure and sequences with features reported and those identified in this study. (B) Identification of PCR bands of cloned CD44 transcripts in human epidermis. PCR bands numbers corresponding to CD44 transcripts are indicated. (C) Analysis of PCR products of skin and epidermal cDNA in acrylamide gel and comparison of sizes of native CD44 transcripts with cloned CD44 transcripts. Transcripts with * are missing some sequences. Previously unreported forms are shown in bold characters. (D) Analysis of CD44 transcripts in human skin obtained from subjects with different anatomical location, gender, age and ethnicity. Samples were taken from: 1, Japanese, female, face, 97; 2, Japanese, male, finger, 71; 3, non-Japanese Asian, male, 24, location unknown.
Table 2.
Characteristics of the 18 CD44 transcripts in human epidermis.
Fig 5.
Protein expression induced by cloned CD44 transcripts.
(A) Immunofluorescence analysis of stable protein expression by cloned CD44 transcripts in transfected CHO-K1 cells. The expressed proteins were detected with an antibody to His-tag. Scale bars, 200 μm. (B) Clones 3 and 11 had stop codons before His-tag.
Fig 6.
Analysis of regulation of variant CD44 transcript expression by various agents in NHK cultures.
(A) After low to high calcium switch with or without FCS, cells were cultured for the indicated number of days. With exception of day 0, all cells were cultured in high calcium medium. (B) NHKs were cultured in high calcium medium with or without various agents. H2O2 = hydrogen peroxide. (C) NHKs were cultured in low or high calcium medium with or without selected agents. All experiments were performed 3 times and representative results are shown.
Fig 7.
Analysis of CD44 transcripts in several cell types.
(A) CD44 transcripts in different cells. (B) Experimental design for analysis of regulation of CD44 expression in normal and malignant keratinocytes by FCS. Cells were treated and harvested at the indicated time points. Numbers in parenthesis correspond to the lane numbers in C and D below. (C, D) Analysis of CD44 transcripts in DJM-1 (C) and NHK (D) cells cultured with or without FCS containing or not CaCl2. All experiments were performed 3 times and representative results are shown.