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Fig. 2. Laminin 5 increases the expression of E-cadherin in HT-29 cells. (A) Upper panel: western blot analysis of the total amount of E-cadherin during the time course of HT-29 cell culture onto laminin 5 matrix. Expression of actin was used as a loading control. Lower panel: the band densitometry was normalized with actin to allow quantification. Data indicate a significant increase in E-cadherin expression during the first 24 hours of culture. Data represent mean ± s.e.m. of three independent experiments. ^*P<0.05; ^**P<0.01, as determined by Student's t-test. (B) Upper panel: western blot analysis of the total amount of E-cadherin after 24 hours of culture onto various concentrations of purified laminin 5. Expression of actin was used as a loading control. Lower panel: the band densitometry was normalized with actin to allow quantification. Data indicate a dose-dependent induction of E-cadherin expression. Data represent mean ± s.e.m. of three independent experiments. ^**P<0.01, as determined by Student's t-test. (C) ECM proteins differentially increase expression of E-cadherin. HT-29 cells were grown for various times (0-24 hours) on plastic (PL) or different ECM proteins used at 10 µg/ml (FN, fibronectin; CO IV, collagen type IV; LN 5, laminin 5). Cell lysates were assayed for E-cadherin expression, using actin as an internal control. The density ratio of E-cadherin:actin is expressed as a relative factor of the value obtained from HT-29 cells in suspension (mean ± s.e.m. of three independent experiments). (D) Increase in E-cadherin expression is mediated by laminin 5-integrin interactions. Western blot analysis of the total amount of E-cadherin in HT-29 cells cultured for 24 hours on plastic (PL) or 5 µg/ml purified laminin 5 (LN5) in the presence of mAbs directed against integrin subunits at a concentration of 10 µg/ml: P4C10 (anti-ß₁), ASC8 (anti-ß₄), BHA2.1 (anti-₂), P1B5 (anti-₃) and GoH3 (anti-₆) or laminin 5 (₃ chain), with a 1/10 diluted BM165 mAb (from cell culture supernatant). Expression of actin was used as a control. Band densitometry was normalized with actin to allow quantification and the % of control (i.e. laminin 5-induced E-cadherin expression without mAbs) was reported on the histogram. Data represent mean ± s.e.m. of three independent experiments. ^*P<0.05; ^**P<0.01, as determined by Student's t-test. Laminin 5-mediated increase in E-cadherin expression was dependent on ₆ß₄ and ₃ß₁ integrins.

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