Expression of BPAG1 in normal human melanocytes and human melanoma cell lines.

<p>(A) The expression of BPAG1 and BPAG2 mRNA was quantified by RT-PCR in normal human melanocytes (NHM) and human melanoma cell lines A375 and G361. Normal human keratinocyte (NHK) mRNA was used as a positive control. β-actin was amplified as a loading control for cDNA. NTC; no template control. (B) The expression of BPAG1 protein was detected by IP-western blotting in human melanoma cell lines A375 and G361. A431 was used as positive control for BPAG1. The arrow indicates BPAG1.</p

Overview of the rapid method for isolating auto-antibody against tumor-associated antigen (TAA) using a scFv library.

<p>The tumor-homing scFv-presenting phages were collected from tumors that were injected with a scFv library. The collected phages were infected to HB2151 for scFv secretion. The secreted scFvs from HB2151 were transferred to nitrocellulose membranes by colony lift. The membranes were incubated with tumor lysate followed by serum from a tumor-bearing mouse. The scFv-tumor protein complex was detected by auto-antibodies. The complex was digested into peptide by trypsin and analyzed using MALDI-TOF mass spectrometry for identification.</p

Quantification of anti-BPAG1 auto-antibodies in melanoma patients.

<p>(A) The levels of anti-BPAG1 auto-antibodies in sera collected from healthy volunteers and melanoma patients were quantified using a MESACUP BP230 ELISA Kit. The INDEX values were plotted and the average INDEX values as shown (±S.E.M.) for control subjects (1.64±0.27) and melanoma patients (3.47±0.40). (B) The melanoma patients were classified using the American Joint Committee on Cancer (AJCC) 2002 staging criteria. <i>In situ</i>, stage I or stage II patients were categorized as “early”, while stage III or stage IV patients were categorized as “advanced”. The average INDEX values (±S.E.M.) of early and advanced melanoma patients were 4.14±0.83 and 3.15±0.43, respectively; the bars indicate the average INDEX value.</p

Identification of bpag1 as a tumor antigen recognized by auto-antibodies.

<p>(A) An example of the screening output. ScFv-tumor antigen complex was detected with auto-antibodies in tumor-bearing mouse serum. (B) Eight candidates were identified by MALDI-TOF mass spectrometry with statistical significance (p<0.05); expect  =  expectation value. (C) Comparison of bpag1, tbc1d13 and c7orf30 expression in NIH-3T3 cells (white bar), F10 melanoma cells (black bar) and F10 melanoma tumors (grey bar) by SYBR Green real-time PCR.</p

Schematic model for mechanical contraction of sweat glands during sweat secretion.

<p>Tubular secretory portions, but not ducts, of sweat glands have self-entangled coiled structural features. Elongated myoepithelial cells are arranged longitudinally parallel to the entangled secretory tubules. Nerve fibers enwrap myoepithelial cell layers surrounding the secretory portions. In sweat glands, multiple elongated myoepithelial cells synchronously contract their tubular secretory portions for excretion of sweat (upper panel), while stellate-shaped myoepithelial cells individually contract their acinar secretory portions of salivary and mammary glands (lower panel).</p

Molecular profiling of human sweat gland compartments.

<p>(A) Schematic illustration of a human sweat gland and its main tissue segments. (Left) Diagram of the sweat gland structure in human skin; (center) diagram of a sweat gland cross section in human skin; (upper right) diagram of the cellular arrangement in the duct consisting of luminal and basal cells surrounded by basement membrane; (lower right) diagram of the cellular arrangement in the secretory portion consisting of luminal and myoepithelial cells surrounded by basement membrane. (B) Cross section of sweat gland coiled tubules in the deep dermis of human skin. HE-stained skin cross section shows the sweat glands surrounded by adipocytes and connective tissue. The coiled regions of sweat glands were detected as rounded structures in the hypodermis. Boxed area in the upper panel is magnified in the lower panel. (C) Expression patterns of sweat gland cell markers K8, αSMA, K77, and S100A2 in human sweat glands. K8 and K77 were expressed in parts of the luminal cell layers, and αSMA and S100A2 in parts of the basal cell layers. Insets show magnified views of the sweat glands. (D) Double immunofluorescence detection of sweat gland markers K8, αSMA, K77, and S100A2 in sweat glands. Insets show magnified views. Nuclei (blue) were counterstained with Hoechst 33342. (B–D) Scale bars: 50 μm.</p

Histological anatomies of basement membranes, cell surface receptors, and cytoskeleton markers in coiled structure compartments of sweat glands.

<p>Expression patterns of (A) LN332 and LAMA5 (basement membrane markers), (B) ITGB1 and ITGA6 (cell surface receptor markers), and (C) E-cadherin and phalloidin (cytoskeleton markers) in the basal layers of sweat glands. Arrowheads and arrows in (B) indicate secretory portions and ducts, respectively. Black arrowheads and arrows in (C) indicate strong detection of actin filaments in ductal apical and secretory basal sides, respectively. Arrowheads and arrows indicate expression of E-cadherin in ductal basal and secretory luminal cell layers, respectively. Double immunofluorescence detection of LN332 and LAMA5 (D), and ITGB1 and ITGA6 (E) with sweat gland basal cell markers S100A2 and αSMA, and E-cadherin and phalloidin (F) with sweat gland secretory portion markers K8 and αSMA in sweat gland cross sections. (G) Expression patterns of CD31 (blood vessel) and PGP9.5 (nerve fiber) markers in sweat glands. Sweat glands boxed in the upper panels are shown in the lower panels at higher magnification. Nuclei (blue) were counterstained with Hoechst 33342. Scale bars: 50 μm.</p

Imaging of the 3D structure of sweat glands.

<p>(A) Neutral red-positive sweat gland coiled organs detected in human skin tissue. The boxed region is shown at higher magnification in the left panel. (B) Visualization by whole-mount staining of an entire sweat gland embedded in human skin. αSMA and S100A2 were detected in sweat glands by double immunofluorescence. Asterisks and double asterisks indicate αSMA-positive secretory portions and S100A2-positive coiled ducts, respectively. Arrowheads and arrows indicate hair follicles and blood vessels, respectively. The dashed line indicates the skin surface. (C) Procedure for whole-mount staining of coiled fragments of sweat glands. The left panel shows neutral red-stained sweat gland organs collected from human skin tissue. (D) Visualization of the basement membrane of the entire sweat gland coiled structure by whole-mount staining for LN332. (E) Double immunofluorescence detection of LN332 and LAMA5 in sweat glands. (F) Imaging of ductal and secretory portions of coiled fragments dissected from sweat glands. Double immunofluorescence detection of K8 and S100A2 in an isolated sweat gland. (Left panel) Projection image of a whole-mount 3D sweat gland stained for K8 and S100A2. (Right panel) Optical section of the whole-mount image. Arrows and arrowheads indicate S100A2-positive ductal and K8-positive secretory portions, respectively. Nuclei (blue) were counterstained with Hoechst 33342 (B, E, and F). Boxed areas in the left panels are shown at higher magnification in the right panels (D, E). Scale bars: 200 μm (A) and 50 μm (B, D–F).</p

Imaging of sweat gland blood vessels and nerve fibers.

<p>(A) Immunofluorescence detection of CD31 and phalloidin in sweat glands. Arrows and arrowheads indicate secretory portions and ducts of sweat gland tubules, respectively. (B) Double immunofluorescence detection of CD31 and LN332 in sweat glands. Arrowheads indicate blood vessels running parallel to sweat gland tubules. (C) Immunofluorescence detection of PGP9.5 in sweat glands. Arrows indicate nerve fibers wrapping around the tubules of sweat glands. Arrowheads indicate ductal tubules. (D) Immunofluorescence detection of PGP9.5 and αSMA in sweat glands. Lower panel shows optical sections of sweat glands. Arrows indicate nerve fibers that enwrap the αSMA-positive secretory portions. The boxed area in the upper panel is shown at higher magnification in the lower panel (A–C). Nuclei (blue) were counterstained with Hoechst 33342. Scale bars: 50 μm.</p