Expression of the 90K Tumor-Associated Protein in Benign and Malignant Melanocytic Lesions  Anna Maria Cesinaro, Gian Paolo Trentini, Dr.  Journal of Investigative Dermatology  Volume 119, Issue 1, Pages 187-190 (July 2002) DOI: 10.1046/j.1523-1747.2002.17642.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Immunohistochemical staining profile for 90K. Sections from paraformaldehyde-fixed and paraffin-embedded tissues were immersed in ethylenediamine tetraacetic acid buffer at pH 6.5 and pretreated in a microwave oven at 360 W for 30 min; endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 10 min at room temperature; sections were incubated with anti-90K monoclonal antibody 1A4.22 (Tinari et al, 1997) for 30 min at room temperature, then were treated with the LSAB Plus System (streptavidin–biotin, LabVision, Fremont, CA); diamino-benzidine was used as chromogene; sections were counterstained with hematoxylin for 2 min. A positive control (breast carcinoma) and a negative control for each case studied were included. Negative normal melanocytes at the dermo-epidermal junction (A) and negative cells in compound nevus (B); positive melanoma cells compared with negative nevus cell in a case of melanoma arisen in nevus (C); strong positivity in “in situ” melanoma (D) and in invasive melanoma (E); the same invasive melanoma as negative control after incubation without the primary antibody (F); moderate positivity in Spitz's nevus (G) and in metastasizing Spitz's nevus (H); positive cutaneous (I) and lymph node (L) metastases. Scale bar: 100 µm. Journal of Investigative Dermatology 2002 119, 187-190DOI: (10.1046/j.1523-1747.2002.17642.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Cell adhesion to 90K protein. (A) Cells from the human melanoma cell line MEL 8863 were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% (vol/vol) penicillin (100 U per ml), 1% (vol/vol) streptomycin (100 U per ml), and 1% (vol/vol) L-glutamine (all from Gibco-BRL, Grand Island, NY), maintained at 37°C in 5% CO2 and passaged every week. Cells (50,000 cells per well) were seeded in 96-well microtiter plates and coated with 100 µl of 90K, laminin, and fibronectin (10 µg per ml in phosphate-buffered saline) overnight at 4°C. One hour before seeding, plates were saturated with 1% bovine serum albumin in RPMI at 37°C. Cells were trypsinized, washed, and resuspended at 500,000 per ml in RPMI serum free with 0.1% bovine serum albumin. One hundred microliters (50,000 cells) were seeded in the wells and, after 1 h of incubation at 37°C, nonadherent cells were removed by gentle washing in phosphate-buffered saline and adherent cells fixed with 95° ethanol for 10 min and stained with 100 µl of 4% crystal violet for 30 min. The absorbance at 550 nm was measured on a plate reader after solubilization with 50 µl of 0.25% Triton-X100. Data represent the mean ± SD of three different experiments done in quadruplicate. (B) Adhesion assay was performed seeding MEL 8863 cells in 96-well microtiter plates precoated with 100 µl of 90K (10 µg per ml) in presence of the function blocking anti-β1 antibody 4B4 (Coulter, Hialeah, FL), anti-90K SP-2 and 1A4.22 antibodies (Tinari et al, 1997) at a concentration of 10 µg per ml, anti-galectin 3M3/38 antibody, or 50 mM lactose. Anti-galectin-3 and lactose did not affect the binding of MEL 8863 cells to 90K, as well as 1A4.22 antibody, whereas adhesion was competed by the anti-90K antibody SP-2; moreover, β1 function-blocking antibody 4B4 markedly reduced the binding of cells to 90K. Data represent the mean ± SD of three different experiments done in quadruplicate. Journal of Investigative Dermatology 2002 119, 187-190DOI: (10.1046/j.1523-1747.2002.17642.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions