Tiziana De Angelis, Adele Noè, Mitali Chatterjee, Joy Mulholland

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Stromelysin-1 Activation Correlates with Invasiveness in Squamous Cell Carcinoma1 Tiziana De Angelis, Adele Noè, Mitali Chatterjee, Joy Mulholland Journal of Investigative Dermatology Volume 118, Issue 5, Pages 759-766 (May 2002) DOI: 10.1046/j.1523-1747.2002.01755.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Effect of batimastat on cell invasiveness and motility. Cells were plated on uncoated or Matrigel-coated porous filters. After a 24 h incubation, the cells were stained and the number of cells passing through the filters was determined. Each assay was performed in triplicate. Mean and SD for five assays of each cell line for untreated cells, two assays for 0.1 µM batimastat (BB-94), and four assays at 1.0 µM batimastat. (a) Number of cells passing through uncoated filters. For untreated SCC cells compared with untreated HaCaT, p = 0.07 for SiHa, 0.04 for FaDu*, 0.06 for A431. For treatment with 1.0 µM batimastat compared with untreated cells, p = 0.03 for HaCaT**, 0.29 for SiHa, 0.15 for FaDu, 0.13 for A431. *Statistically significant (p < 0.05) difference compared with untreated HaCaT; **statistically significant difference for batimastat-treated compared with untreated cells of the same cell line. (b) Invasion of Matrigel-coated filters. Untreated SCC versus untreated HaCat, p = 0.20 for SiHa, 0.02 for FaDu*, 0.10 for A431. Batimastat at 1.0 µM compared with untreated cells, p = 0.40 for HaCaT, 0.09 for SiHa, 0.02 for FaDu**, 0.09 for A431. Journal of Investigative Dermatology 2002 118, 759-766DOI: (10.1046/j.1523-1747.2002.01755.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Steady-state levels of mRNA for metalloproteinases and TIMPs. Mean and SD for two RT-PCR reactions for each mRNA. (a) Matrilysin and stromelysins 1, 2, and 3; (b) TIMP-1; (c) TIMP-3. Journal of Investigative Dermatology 2002 118, 759-766DOI: (10.1046/j.1523-1747.2002.01755.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Immunoblots of metalloproteinases and TIMP in conditioned medium. Culture medium from each cell line was collected after 48 h incubation. Expression of specific proteins was analyzed by gel electrophoresis and immunoblotting. Approximate molecular weights listed were determined by comparison with molecular weight standards. MMP2 = gelatinase A, 66 kDa; MMP3 = stromelysin-1, 59 kDa and 48 kDa; MMP7p = promatrilysin, 28 kDa; MMP7a = active matrilysin, 38 kDa aggregate; MMP9 = gelatinase B, 83 kDa; MMP10 = stromelysin-2, 50 kDa; MMP11 = stromelysin-3, 47 kDa. TIMP1 = 28.5 kDa. Journal of Investigative Dermatology 2002 118, 759-766DOI: (10.1046/j.1523-1747.2002.01755.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Immunocytochemistry for active matrilysin. All cells were fixed 24 h after removal of aphidicolin. Arrowheads designate mitotic cells. (a) HaCaT; (b) SiHa; (c) FaDu; (d)A431. Scale bar: 100 µm. Journal of Investigative Dermatology 2002 118, 759-766DOI: (10.1046/j.1523-1747.2002.01755.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Immunocytochemistry for stromelysin- 1. All cells were fixed 24 h after removal of aphidicolin. Arrowheads designate mitotic cells. (a) HaCaT; (b) SiHa; (c) FaDu; (d) A431. Scale bar: 100 µm. Journal of Investigative Dermatology 2002 118, 759-766DOI: (10.1046/j.1523-1747.2002.01755.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Immunocytochemistry for TIMP1. All cells were fixed 24 h after removal of aphidicolin. Arrowheads designate mitotic cells. (a) HaCaT, large cell at top of figure is binucleate; (b) SiHa; (c) FaDu; (d) A431. Arrowhead at bottom of figure designates daughter cells in cytokinesis. Scale bar: 100 µm. Journal of Investigative Dermatology 2002 118, 759-766DOI: (10.1046/j.1523-1747.2002.01755.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions