JCS018440 Supplementary Material

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• Supplemental Table S1 -

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• Supplemental Figure S1 -

  Fig. S1. Generation and characterisation of β1-integrin-GFP cells. (A) FACS trace of MEF 7929 (floxed β1^+/+) and MEF 7929 (β1^−/−) cells. β1 integrin was detected with KMI6 rat anti-mouse β1 integrin. (B) PCR of MEF 7929 (β1^+/+), MEF 7929 (β1^−/−) and MEF 4043 (with wild-type β1). The top panel shows PCR for β1 integrin, and the lower panel shows PCR of the Immorto allele. (C) Western blot of MEF 7929 (floxed β1^+/+) and MEF 7929 (β1^−/−) cells using KMI6 or rat IgG. (D) Ablation of β1 integrin expression prevented attachment to the α2β1 ligand, type I collagen. MEF 7929 carrying floxed β1 integrin attached normally to collagen I. MEF 7929 (β1^−/−) cells were still able to attach to the 50 kDa fragment of fibronectin, most likely via αVβ3 integrin. (E) FACS trace of MEF 7929 (β1^−/−) cells retrovirally transduced with GFP-pFBneo or β1-GFP-pFBneo. (F) Attachment of MEF 7929 (β1^−/−) cells retrovirally transduced with GFP-pFBneo or β1-GFP-pFBneo to fibronectin. Both cell lines attached to fibronectin in a dose-dependent manner. Attachment was cation-dependent (ablated by 20mM EDTA) and was reduced by a combination of function-blocking rat anti-mouse β3 integrin (2C9.G2) and rat anti-human β1 (mAb13) antibodies (10 µg/ml each). (G) Expression of GFP-tagged β1 integrin restores β1 KO MEF attachment to laminin-1 and collagen-1. Attachment to laminin-1 and collagen-1 was inhibited by rat anti-mouse α6 integrin antibody GoH3 and rat anti-mouse α2 antibody HMa2, respectively (10 µg/ml each). (H) GFP-transfected MEF 7929 (β1^−/−) (top two panels) and β1-GFP-transfected MEF (bottom two panels) spread and formed adhesion contacts on fibronectin. Cells were co-stained with either TRITC-phalloidin for F-actin or anti-vinculin. Merged images are shown in far right panels. Scale bar: 20 µm.

• Supplemental Figure S2 -

  Fig. S2. S976162 induces LIBS and reduces LABS epitope expression in a dose-dependent manner. (A) Binding of the mouse anti-human β1 integrin monoclonal antibody 12G10 which detects a ligand-induced binding site (LIBS) epitope to affinity-purified α4β1 in the presence of varying concentrations of S976162 (open circles), or equivalent dilutions of dimethylformamide solvent (DMF; filled circles). (B) Binding of the rat anti-human β1 antibody mAb13 which detects a ligand-attenuated binding site (LABS) epitope. Assays were performed as described by Newham et al. (Newham et al., 1998). Briefly, purified α4β1 was immobilised and BSA blocked in a 96-well ELISA plate format before incubation for 2 hours at 37°C with 0.1 µg/ml 12G10 in the presence of compounds or DMF, or 0.3 µg/ml mAb13 in the presence of 2 mM Mn²⁺ and small molecule inhibitors or DMF. Bound antibody was detected with anti-rat HRP and the substrate ABTS as previously described.

• Supplemental Figure S3 -

  Fig. S3. Demonstration of biexponential analysis of FRET data. (A) Lifetime map example of β1 integrin:talin association in cells plated on finronectin (as in Fig. 1). The yellow box depicts the focal adhesion subsequently masked and used for biexponential analysis of FRET poplulations. (B) Data were analysed using biexponential fit (as described in the Materials and Methods) and the graph of fit is shown along with associated residuals, the two lifetime population values and the Chi-squared value to demonstrate goodness of fit.