Figure 1.
Deposition of Lm332 on culture plates by normal keratinocytes (NHK) and five cancer cell lines (A431, CaSki, HSC-4, STKM-1 and MKN-45).
Cells were suspended in serum-free medium, inoculated at a density of 5×106 cells per 90-mm dish, and incubated for 2 days. The resulting ECM and CM were prepared from each culture. To prepare ECM, cells were removed from the dishes by treating them with 10 mM EDTA and briefly with 20 mM NH4 OH and then washing with PBS. The ECMs on the dishes were extracted with the SDS sample buffer. A twentieth part of the CM and a thirtieth part of the ECM were subjected to immunoblotting with the antibodies to the laminin α3, γ2 and ß3 chains under reducing conditions. Bars indicate the position and size of the laminin chains. Other experimental conditions are described in “Materials and Methods”.
Figure 2.
Immunofluorescent staining of Lm332 deposited by three cell lines.
NHK (A, top), HSC-4 cells (A, center) and Lm332-HEK cells (A. bottom) were suspended in serum-free medium, inoculated at a cell density of 2×103 cells/well on collagen-coated 8-well chamber slides and incubated for 6 h. The cultures were stained for F-actin with rhodamine phalloidin (left panels) and for Lm332 with the anti-α3 chain BG5 antibody and followed by a FITC-labeled secondary antibody (center panels), as described in “Materials and Methods”. Right panels are merged images. In (B), Lm332-HEK cells were inoculated at a high cell density (1×105 cells/well), incubated for 6 h (left panel) or 48 h (right panel), and stained for Lm332 as above.
Figure 3.
Effect of sodium selenate on Lm332 deposition by NHK cells.
(A) Immunoblotting analysis. NHK cells were inoculated in serum-free medium at a density of 4×105 cells per 35-mm dish, incubated overnight, and treated with (+) or without (−) 0.1 mM sodium selenate (Sigma) at 37°C for 24 h. After the incubation, the ECM and CM were prepared from each culture and analyzed for the laminin α3 chain by immunoblotting as described in Figure 1. (B) The ECMs from the control (none) and selenate-treated cultures (+selenate) were subjected to immunofluorescence staining with the anti-laminin α3 chain antibody BG5. (C) Phase-contrast micrographs of control and selenate-treated cultures. Other experimental conditions are described in “Materials and Methods”.
Figure 4.
SDS-PAGE analyses of Lm332-ECM deposited by Lm332-HEK cells.
Confluent cultures of Lm332-HEK (lane 2), α3AA-Lm332-HEK (lane 3) and ß3γ2-HEK (lane 4) were incubated in the serum-containing growth medium for 4 days, and the resultant ECMs were prepared and applied to SDS-PAGE under reducing conditions, followed by CBB staining (A) or immunoblotting with anti-α3 (B, upper panel) and -γ2 (B, lower panel) chain antibodies. Purified Lm332 was run as a standard on lane 1. Bars on the left indicate major protein bands with their approximate molecular sizes in kDa. The two minor bands (# and *) in (A) were identified as laminin γ2 fragments by NH2-terminal amino acid sequencing. Other experimental conditions are described in “Materials and Methods”. (C) To see the proteolytic processing of deposited Lm332, Lm332-HEK cells were incubated for 6 h and 30 h, and the deposited Lm332 was prepared and applied to SDS-PAGE under reducing conditions, followed by immunoblotting for the α3 chain (left panels) and the γ2 chain (right panels). (D) CM (lane 1) and ECM (lane 2) were prepared from the confluent culture of Lm332-HEK cells after incubation in serum-free medium for 2 days and analyzed by immunoblotting under non-reducing conditions with anti-α3 (left panel) and -ß3 (right panel) chain antibodies. Lane 3, purified Lm332 with processed γ2 chain (400 kDa). Note that Lm332 in the ECM remains on the gel top as a broad band (lane 2).
Figure 5.
Fine structures of Lm332-ECM (left), ß3γ2-ECM (center) and purified Lm332 (right) were analyzed by TEM at a magnification of × 50,000.
Bars indicate 100 nm.
Figure 6.
Migration and morphology of NHK cells on purified Lm332 and Lm332-ECM substrates.
(A) NHK cells were inoculated on Lm332-coated plates (left three columns) or deposited ECMs (right four columns), which were prepared as described below. After 1.5 h incubation, cell migration was monitored by video microscopy for 5.5 h. Purified Lm332 was coated at a concentration of 1.0 or 2.5 µg/ml on a non-treated plate, or at 1.0 µg/ml on a plate pre-coated with the anti-laminin α3 chain antibody LSαc3 (Ab). ECM substrates were prepared from Lm332-HEK (WT), α3AA-Lm332-HEK (AA), and ß3γ2-HEK (ßγ) cultures. In the right two columns, ß3γ2-ECM and Lm332-ECM were further coated with 1.0 µg/ml purified Lm332 (+Lm332) and then used for the migration assay. On ß3γ2-ECM alone or a non-treated plate, NHK cells never attached and migrated during the initial 7 h (data not shown). Each bar represents the mean ± S.D. of the migration speeds of 8 cells in each assay. These results were essentially reproduced in three independent experiments. Other experimental conditions are described in “Materials and Methods”. (B) CM and deposited ECM (ECM) were prepared from confluent cultures of NHK cells. The CM was coated on a 24-well plate. Migration of NHK cells on these substrates was analyzed as above. (C) NHK cells were incubated for 3 h on ß3γ2-ECM, Lm332-ECM and plates coated with 1.0 or 2.5 µg/ml purified Lm332, and cell morphology was observed under a phase-contrast microscope. Original magnification, ×300. (D) In the same cultures as (C), actin cytoskeleton was visualized with FITC-phalloidin. Numerical values in parentheses indicate the concentration (µg/ml) of Lm332 coated.
Figure 7.
Cell adhesion activity of Lm332-ECM and purified Lm332 toward NHK cells.
ECMs from ß3γ2-HEK and Lm332-HEK cultures and Lm332-coated plates were prepared as described in Figure 4. (A) Phase-contrast microscopic images of NHK cells after 10 min incubation. Original magnification, ×300. Lm332 protein was coated at 1.0 µg/ml on the plate. (B) NHK cells suspended in KGM growth medium were inoculated into each well and then incubated at 37°C for 10 min. After the incubation, non-adherent cells were removed, and adherent cells were quantified. Numerical values under three right columns indicate the concentration at µg/ml of coated Lm332 protein. Each bar represents the mean ± S.D. of the fluorescent intensity (FI) for adherent cells in triplicate assays. The data shown are representative of at least three independent experiments performed.
Figure 8.
Interaction of purified Lm332 or Lm332-ECM with integrins.
(A and B) To identify integrins responsible for cell adhesion, NHK cells suspended in the KGM medium were pretreated with the indicated integrin antibodies (2 µg/ml IgG) or EDTA for 30 min at room temperature and plated on the plates with purified Lm332 (A) or Lm332-ECM (B). After 20 min incubation, adherent cells were quantified. The relative number of adherent cells in the presence of control mouse or rat IgG was taken as 100%. Each bar represents the mean ± S.D. of the fluorescent intensity (FI) for adherent cells in triplicate assays. (C) Morphology of NHK cells was examined after incubation on Lm332-ECM in the presence of control mouse IgG, or the indicated anti-integrin neutral antibodies for 20 min. In the upper left culture (Lm332+IgG), NHK cells were incubated in a well precoated with 1.0 µg/ml purified Lm332 in the presence of the control IgG. Original magnification, ×300. The images shown are representative of at least three independent experiments performed. (D) Binding affinity of integrin α3ß1 to purified Lm332 and Lm332-ECM was determined by ELISA. Varied concentrations of purified integrin α3ß1 was allowed to bind to the plates coated with 1 µg/ml purified Lm332 (closed squares) or deposited with Lm332-ECM (open circles) in the presence of 1 mM MnCl2. Bound integrin α3ß1 was quantified by ELISA using an anti-integrin α3ß1 polyclonal antibody. The amounts of integrin α3ß1 bound in the presence of 10 mM EDTA was taken as nonspecific binding and subtracted as the background. The results shown are the means of duplicate assays.
Figure 9.
Differential detachment of NHK cells adhered to purified Lm332 and Lm332-ECM.
NHK cells were inoculated onto the plates coated with 1 µg/ml purified Lm332 or deposited with Lm332-ECM and incubated for 1 h. After washing with PBS, the cells were incubated with trypsin/EDTA diluted 1∶35 in PBS (A) or with 10 mM EDTA alone (B). After incubation for the indicated lengths of time and then washing with PBS, the relative number of adherent cells was determined as described in Figure 5. Each bar indicates the mean ± S.D. of the fluorescent intensity (FI) for adherent cells in triplicate assays.
Figure 10.
Localization of integrin ß4 in NHK cells on purified Lm332 and Lm332-ECM.
Glass-bottom dishes (Asahi Techno Glass, Tokyo, Japan) were previously coated with 2.0 µg/ml Lm332 or deposited with Lm332-ECM. NHK cells were inoculated and incubated in growth media. After incubation for 5 h, the cells were washed with PBS and then fixed with 4% (w/v) paraformaldehyde in PBS for 10 min and then treated without (w/o Triton) or with (Triton) 0.5% (v/v) Triton X-100. The fixed cells were stained with an integrin ß4 antibody and an Alexa Fluor 488-labeled secondary antibody. Other experimental conditions are described in “ Materials and Methods”.