l adhesion vs. the enhanced cell motility, are caused from the different states of Lm332, i.e. polymerized Lm332 matrix and nonpolymerized soluble Lm332. It is assumed that the soluble or unassembled form of Lm332 plays an important role in the elevated cell migration during the wound healing and tumor invasion. Materials and Methods Antibodies and Reagents Lm332. The strong and stable cell adhesion to Lm332-ECM, which was evident by the resistance of keratinocytes to cell detachment treatments, lead to the suppressed cell migration. Both Lm332-ECM and 3c2-ECM hardly supported cell migration, but treatment of these ECMs with purified Lm332 enhanced cell migration. Even in this experiment, the Lm332-ECM coated with purified Lm332 showed suppressed cell motility activity as compared with purified Lm332 alone or 3c2-ECM plus Lm332. These results clearly indicate that Lm332-ECM rather inhibits cell migration. The strong cell adhesion to Lm332-ECM obviously depends on the interaction with integrins. It has been reported that keratinocytes interact with self-deposited Lm332 through integrins a31 for polarization and migration. AT 7867 manufacturer integrin a31 also functions for the proper organization of deposited Lm332. Our results indicated that integrin a31 bound to Lm332-ECM in a much higher affinity than purified Lm332. In addition, experiments with neutral integrin antibodies suggested that Characterization of Polymerized Laminin-332 Matrix type VII collagen antibody from Sigma, anti-type IV collagen antibody from Santa Cruz, and antiperlecan antibody and Alexa Fluor 488-labelled secondary antibody from Invitrogen. Human recombinant Lm332 and Lm311 were purified as described previously. Human recombinant Lm511 was purchased from BioLamina. Cells and Transfectants Human embryonic kidney cell line HEK293 was purchased from American Type Culture Collection and transfected with the cDNAs of the three Lm332 subunits to overexpress the wild-type Lm332 or an a3mutated Lm332 resistant to the proteolytic processing of the a3 chain . Lm332-producing human cancer cell lines used were epidermoid carcinoma of the vulva, epidermoid carcinoma of the cervix, squamous adenocarcinoma of the tongue and gastric adenocarcinomas. STKM-1 was established and provided by Dr. S. Yanoma , and the others were obtained from Japanese Cancer Resources Bank. Expression of Lm332 in these cancer cell lines were reported in our past studies. All these cell lines were stored in a liquid N2 tank in our laboratory and cultured in DMEM/F12 medium supplemented with 10% fetal calf serum, penicillin and streptomycin sulfate. NHK cells from neonatal foreskin were obtained from Cascade Biologics, 
and cultured in KGM medium, which was composed of keratinocyte basal medium, 0.1 ng/ml human EGF, 0.4% bovine pituitary extract, 10 mg/ml PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22189542 insulin, 500 ng/ml hydrocortisone, 50 mg/ml gentamicin, and 50 ng/ml amphotericin B. Passages 2 and 3 were used in the described experiments. Preparation of CM, Deposited ECM, and Lm332-coated Plates Unless otherwise noted, cells were grown to subconfluence in the growth medium, washed three times with PBS and then incubated in serum-free medium. Two days later, the CM was collected and added with two protease inhibitors, phenylmethylsulfonyl fluoride and N-ethylmaleimide. The CM was dialyzed against pure water, lyophilized and then dissolved in a 1/50 volume of PBS. To prepare deposited ECM, subconfluent cultures were incubated in the growth medium for 2 days with medium
