suspension in serum-free medium was inoculated per well of the chamber slides and incubated for 6 h. The cultures were washed with PBS, fixed with 4% paraformaldehyde in PBS for 10 min, and then treated with 0.5% Triton X-100 in PBS for 15 min. The fixed cells were blocked with 2% BSA in PBS for 1 h and then reacted with the mouse anti-laminin a3 chain antibody BG5 or other antibodies for 1 h. After washing with PBS, the 
cultures were incubated with a mixture of FITC-conjugated goat anti-mouse IgG antibody and rhodamine phalloidin for 1 h, and then washed with PBS. Fluorescence images were obtained using fluorescence microscope BZ-8000 or a LSM510 confocal microscope. To immunostain Lm332 in the ECMs deposited by various types of cells, the ECMs were prepared as described above and MedChemExpress PHA-793887 directly subjected to the staining without the fixation. In the analysis of integrin localization, rabbit polyclonal antibodies against 4 integrin and a6 integrin were used as primary antibodies. Cell Detachment Assay NHK cells were seeded into each well deposited with Lm332-ECM or coated with purified Lm332 in 96-well plates, and incubated for 1 h at 37uC. The cells were then treated with a solution of trypsin/EDTA diluted 1:35 in PBS or with 10 mM EDTA/PBS for varied lengths of time. The relative number of adherent cells was determined as described in the cell adhesion assay section. Transmission Electron Microscopy of ECM Proteins Confluent cultures of Lm332-HEK and 3c2-HEK cells were incubated on poly-L-lysine-coated cover slide glasses for 4 days, and their deposited ECMs were prepared as described above. The ECMs were then fixed with 2% glutaraldehyde and then with osmium tetraoxide. The materials were analyzed with JEOL JEM 200EX at Hanaichi Electron Microscope Technology Institute. Cell Adhesion Assay Cell adhesion assay with NHK cells was performed as described previously. Briefly, each well of 96-well ELISA plates was coated with a substrate protein at indicated concentrations at 4uC overnight and then blocked with 1% BSA. Cells were inoculated per well containing KGM medium, and incubated in described conditions. After nonadherent cells were removed, adherent cells were fixed and stained with Hoechst 333432. The fluorescent intensity of each well of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22187495 the plates was measured using a CytoFluor 2350 fluorometer. For inhibition assay, the cell suspension was incubated with function-blocking anti-integrin Supporting Information deposited by normal keratinocytes and four cancer cell lines. Each kind of cells were inoculated per well of LabTek 8-well chamber slides in serum-free medium and incubated Characterization of Polymerized Laminin-332 Matrix for 2 days. After the cells were removed by treating with 10 mM EDTA, deposited Lm332 matrices were immunostained with the anti-laminin a3 chain antibody BG5 and a FITC-conjugated secondary antibody. Other experimental conditions are described in ��Materials and Methods”. Bars, 100 mm. purified Lm332 was determined to be 1.1 by the NIH image software. culture plates by Lm332-HEK cells by ELISA and CBB staining. Fifty ml of purified Lm332 protein were coated at the indicated concentrations to the 96-well plates. Lm332-HEK transfectant was cultured in DMEM/F12 medium supplemented with 10% fetal calf serum, and ECM proteins were deposited on the plates for 3 days. The amount of Lm332 on the plates was determined by ELISA using the antibodies against the laminin a3 and c2 chains. Each bar represents the mean 6 S.D.
