Volume 115, Issue 2, Pages 433-442 (August 1998) Knockout of α6β1-integrin expression reverses the transformed phenotype of hepatocarcinoma cells  Vinicio Carloni*, Roberto G. Romanelli*, Arthur M. Mercurio‡, Massimo Pinzani*, Giacomo Laffi*, Giorgio Cotrozzi*, Paolo Gentilini*  Gastroenterology  Volume 115, Issue 2, Pages 433-442 (August 1998) DOI: 10.1016/S0016-5085(98)70210-0 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Flow-cytometric analysis of integrin subunit expression in HepG2 cells. HepG2 cells were analyzed by flow cytometry using MAbs specific for the indicated integrin subunits. HepG2 cells were incubated with a nonspecific IgG as control (left peak) or antibodies specific for the indicated integrin subunit (right peak). The y axes represent cell number, and the x axes represent fluorescent intensity. Representative experiment of two. Gastroenterology 1998 115, 433-442DOI: (10.1016/S0016-5085(98)70210-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Effect of MAbs on adhesion of HepG2 cells to laminin 1 and collagen I. HepG2 cells were incubated in the presence of antibodies to specific integrin subunits and assayed for their ability to adhere on 10 μg/mL laminin 1 (●) and 10 μg/mL collagen I (▩). Cell adhesion was quantitated after 60 minutes, and values shown are expressed as the percentage of cells bound to poly-L-lysine as described in Materials and Methods. Each bar represents three separate determinations with quadruplicate wells in each experiment. Values are expressed as means ± SD. Gastroenterology 1998 115, 433-442DOI: (10.1016/S0016-5085(98)70210-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Expression levels of the mutant β4 (β4-Δcyt) and α6 integrin subunits in transfected HepG2 cells. Two populations of HepG2 cells transfected with the β4-Δcyt cDNA (clones B4D1 and B4D) were stained using UM-A9, an MAb specific for the β4 integrin subunit, or GoH3, an MAb specific for the α6 integrin subunit. The cells were analyzed by flow cytometry: the left peak in each panel was obtained by using a nonspecific IgG, and the right peak corresponds to the expression levels of the indicated integrin subunit. Representative experiment of two. Gastroenterology 1998 115, 433-442DOI: (10.1016/S0016-5085(98)70210-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Immunoprecipitation analysis of the β4-Δcyt integrin subunit expressed in transfected cells. Transfected cells were surface labeled with 125I and extracted with lysis buffer. Aliquots of surface-iodinated cell lysates, normalized for counts per minute, were then incubated with specific integrin antibodies. Immunoprecipitates were then resolved by 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis under nonreducing conditions. The mock transfectants (HepG2-neo) were immunoprecipitated with anti-β4 antibody (UM-A9) (lane 1), anti-α6 antibody (GoH3) (lane 2), and anti-β1 antibody (AIIB2) (lane 3). The β4-Δcyt transfectants (B4D) were immunoprecipitated with UM-A9 (lane 4), GoH3 (lane 5), and AIIB2 (lane 6). The migration positions of β1 and β4-Δcyt subunits are shown on the right margin. The molecular weight markers are indicated on the left margin. Representative experiment of two. Gastroenterology 1998 115, 433-442DOI: (10.1016/S0016-5085(98)70210-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Adhesion of the HepG2 transfectants on ECM proteins. The β4-Δcyt transfectants (clones B4D1 and B4D) and mock transfectants (HepG2-neo) were plated for 60 minutes at 37°C on microtiter wells coated with 10 μg/mL laminin 1 (▩), 10 μg/mL laminin 2 (■), 10 μg/mL collagen type I (●), and 10 μg/mL laminin P1 fragment (□). The extent of cell adhesion was determined by a colorimetric assay, and the values are reported as the percent of cells bound to poly-L-lysine as described in Materials and Methods. Each bar represents three separate determinations with quadruplicate wells in each experiment. Values are expressed as means ± SD. Gastroenterology 1998 115, 433-442DOI: (10.1016/S0016-5085(98)70210-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Migration of the HepG2 transfectants. Cell migration was measured by the Boyden chamber assay as described in Materials and Methods. HepG2-neo (▩; mock transfectants) and the β4-Δcyt–transfected cells (B4D1 [□] and B4D [■]) were seeded in serum-free DMEM in the upper compartment of the Boyden chamber and tested for migration in the lower chamber through a filter precoated with collagen type I (20 μg/mL) or laminin 1 (20 μg/mL). Cells that had migrated to the lower surface of the filter after 8 hours were stained and visually quantitated by cell counting. The values (cells migrated per high-power field) are expressed as means ± SD and are from six separate fields counted in three different experiments. HPF, high-power field. Gastroenterology 1998 115, 433-442DOI: (10.1016/S0016-5085(98)70210-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Matrigel invasion of HepG2 transfectants. Matrigel was diluted in DMEM, and a thin layer of Matrigel solution (50 μg/mL) was overlaid on the upper surface of the polycarbonate filter. The Matrigel was allowed to gel by incubating the filters for 30 minutes at 37°C. HSC conditioned medium (HSC-CM), EGF (100 ng/mL), or DMEM were added to the bottom wells of the Boyden chambers as indicated. The HepG2 transfectants (105) were added to the top wells of the chambers and then incubated for 24 hours at 37°C. The cells that reached the lower surface of the filters were fixed, stained, and quantitated as described in Materials and Methods. ▩, HepG2-neo; □, B4D. The values are expressed as the mean ± SD from four different experiments performed in duplicate. *P < 0.01 vs. HepG2-neo. Gastroenterology 1998 115, 433-442DOI: (10.1016/S0016-5085(98)70210-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Growth characteristics of HepG2 transfected cells 4 days after plating on culture dishes. (A) The mock-transfected cells (HepG2-neo) grew as clusters that formed multilayered colonies, (B) whereas the β4-Δcyt transfected cells (B4D) grew as a monolayer of polygonal cells (original magnification 200×). Gastroenterology 1998 115, 433-442DOI: (10.1016/S0016-5085(98)70210-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Growth characteristics of HepG2 transfected cells 4 days after plating on culture dishes. (A) The mock-transfected cells (HepG2-neo) grew as clusters that formed multilayered colonies, (B) whereas the β4-Δcyt transfected cells (B4D) grew as a monolayer of polygonal cells (original magnification 200×). Gastroenterology 1998 115, 433-442DOI: (10.1016/S0016-5085(98)70210-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 9 Growth rate of HepG2 transfected cells. The in vitro growth rate in standard culture conditions of parental cells (HepG2, ▩), mock-transfected (HepG2-neo, ●), and β4-Δcyt transfected cells (B4D, ■) was determined by [3H]thymidine incorporation and by cell counting. (A) Cells (20,000/well) were plated on plastic 12-well dishes in the presence of DMEM containing 5% FBS. The medium was changed every 2 days, and the incorporation of [3H]thymidine was determined at the indicated time points as detailed in Materials and Methods. (B) HepG2-neo (●) or B4D cells (■) (20,000/well) were plated in the presence of DMEM containing 10% or 20% serum (FBS). The medium was changed every 2 days, and the number of cells was determined by counting with a hemocytometer at the time points indicated as detailed in Materials and Methods. The values are expressed as mean ± SD of three different experiments performed in triplicate. *P < 0.05 or higher degree of significance vs. HepG2-neo. Gastroenterology 1998 115, 433-442DOI: (10.1016/S0016-5085(98)70210-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions