Impairment in cell-cell contact formation in FMNL2-/- cells can be rescued by reintroduction of FMNL2-GFP.

<p><b>(A)</b> Live wildtype MCF10A cells expressing mCherry-PA-Rac1 and GFP-α-catenin were imaged over time to visualize adhesion junction formation upon Rac1 activation. Small inset shows mCherry-PA-Rac1 expression. <b>(B)</b> Comparison of cell-cell contact formation dynamics upon PA-Rac1 activation in indicated cell lines. Dashed circles highlight regions of interest. <b>(C)</b> Western blot comparing endogenous FMNL2 and exogenous FMNL2-GFP expression. Antibodies were applied as indicated. <b>(D)</b> Quantification of cell-cell contact establishment after Rac1 activation as in (B) (<i>n</i> = 60 (WT), <i>n</i> = 65 (FMNL2-/-), <i>n</i> = 64 (FMNL2-/- + FMNL2-GFP), pooled from three independent experiments, error bars SD, *p ≤0.05,). <b>(E)</b> Quantification of cell-cell contact formation in cells treated either with control or Cdh1 siRNA (<i>n</i> = 43 (si control), <i>n</i> = 44 (si Cdh1) pooled from three independent experiments, error bars SD, *p≤0.5 calculated by <i>t</i>-test). Western blot demonstrating siRNA efficiency in MCF10A cells in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194716#pone.0194716.s001" target="_blank">S1E Fig</a>. <b>(F)</b> Live FMNL2-/- cells co-expressing FMNL2-GFP or FMNL2-NT-GFP with PA-Rac1 (upper small image) were imaged. Stills show protrusion dynamics and contact formation over time. Zoom image depict magnification of filopodia. Asterisks highlight areas of contact sites. <b>(G)</b> Co-immunoprecipitation of HEK cells expressing GFP or E-Cadherin CT-GFP with flag-tagged FMNL2 derivatives. Flag-tagged proteins were precipitated and bound proteins were detected using GFP-HRP antibody. Please note, E-Cadherin CT-GFP is pulled down only with flag-FMNL2 CT. In immunoprecipitates of cells expressing high levels of flag-FMNL2 CT, a band at 60 kD is observed likely deriving from a truncation product.