FIGURE 1.

The role of extracellular matrix (ECM) proteins in human bronchial epithelial cell (HBEC) behaviour. a) HBECs (5×10⁴) were seeded on non-adherent tissue-culture plastic coated with ECM proteins and attachment was determined at 30 min using CYQUANT GR (ThermoFisher Scientific, Hemel Hempstead, UK) with background fluorescence subtracted. Attachment to collagen IV was greater than to laminin (two-way ANOVA with Holm-Sidak's test for multiple comparisons) and vitronectin-coated wells. The experiment was repeated three times in technical triplicate, including using two independent donor cell cultures. Data are presented on a log scale. b) HBECs were incubated in blocking antibodies against integrins α2, α3, αv, α5 and α6 for 20 min before cells (2×10⁴) were seeded onto non-adherent tissue-culture plastic wells coated with either collagen IV, collagen I or fibronectin. Fluorescence with background subtracted was measured using CYQUANT GR. Blocking integrin α2 led to significantly less HBEC attachment in the collagen IV-coated wells (one-way ANOVA with Holm-Sidak's test for multiple comparisons), but not the collagen I- (p=0.141) or fibronectin-coated wells (p=0.881), compared to control wells. The experiment was repeated three times in technical triplicate (indicated in black, grey and white), including using two independent donor cell cultures. c) HBEC cell number was determined using CYQUANT GR and a standard curve of known cell number at 2 h and 48 h after seeding HBECs (1×10⁴) onto non-adherent tissue-culture plastic coated with ECM proteins. No statistical difference was found between the groups (one-way ANOVA with Holm-Sidak's test for multiple comparisons; p>0.25 for all comparisons). The experiment was repeated three times in technical triplicate, including using two independent donor cell cultures. d) Blocking antibodies were added to wells 2 h after seeding HBECs (5×10³) onto collagen IV- and laminin-coated wells. Fluorescence with background subtracted was recorded at 48 h using CYQUANT GR and compared to a control well without blocking antibody. Significantly less epithelial cell proliferation was observed compared to the control in the presence of integrin β1-blocking antibody (one-way ANOVA with Holm-Sidak's test for multiple comparisons). The experiment was repeated six times in technical triplicate, including using two independent donor cell cultures. e) Integrin blocking antibodies were added to air–liquid interface (ALI) cultures at the point of ALI creation and at every feed thereafter. Trans-epithelial resistance (TEER) as a measure of epithelial integrity was recorded at five timepoints after air-lift (1=0 days, 2=7 days, 3=14 days, 4=21 days and 5=28 days). Blockade of α1 and α3 integrins inhibited trans-epithelial resistance (two-way ANOVA with Holm-Sidak's test for multiple comparisons). The experiment was performed by making three repeated measurements from duplicate wells from two independent donor cell cultures (i.e. four independent wells at each timepoint). Data are presented on a log scale. *: p=0.018; **: p=0.002; ***: p=0.006; ****: p<0.001.