Fig. 4. Small molecule inhibitors induce integrin signalling. (A) A375-SM cells were transfected with 4-integrin-GFP and paxillin-mRFP and plated onto coverslips coated with PLL. Cells were then incubated with K20 or mAb13 antibody-coated beads for 30 minutes, followed by treatment with DMF or small molecule inhibitor as indicated. Cells were then imaged using multiphoton FLIM. Cumulative FRET efficiency data from a masked pre-set region around the bead in ten cells per treatment is shown in the histogram. (B) Histogram demonstrating FRET efficiency of 4-paxillin association at K20 beads over a dose-response range of compounds S976162 or S9916197. Results are means ± s.e.m. (C) β1-integrin-GFP fibroblasts were transfected with talin-rod-mRFP. Cells were then plated onto coverslips coated with PLL and allowed to attach and spread for 1 hour. Cells were then incubated with beads coated with K20 for 30 minutes, and treated with either the vehicle control DMF or V0519 compound, and subsequently imaged using FLIM as before. Cumulative FRET efficiency data from a masked pre-set region around the bead in 12 cells per treatment is shown in the histogram.