Ck666

The Lack antigen was produced and purified by the ‘Recombinant Protein' platform (UMR144, Institut Curie, Paris, France), and Lack peptide (aa 156–173) was synthetized by PolyPeptide Group. Cytochalasin D and nocodazole used from centrosome purification were from Sigma Aldrich. For inhibition of Arp2/3 activity, cells were pretreated with 25 μM CK666 (Tocris Bioscience) for 30 min before being stimulated for indicated time in presence of the drug.

Unless otherwise specified, all chemicals and reagents were purchased from Sigma. C3 transferase was obtained from Cytoskeleton (#CT03), The Arp2/3 inhibitor CK-666 was purchased from Tocris (#3950), resuspended in Dimethyl Sulfoxide at a stock concentration of 100 mM, and aliquoted stocks stored at −80°C.

Primary antibodies used were rabbit anti-mDia1 polyclonal (LifeSpan BioSciences, Seattle, WA), rabbit anti-mDia3 polyclonal (SIGMA-Aldrich, St. Louis, MO), chicken anti-vimentin polyclonal (Millipore, Burlington, MA), rabbit anti-GFP polyclonal (MBL, Nagoya, Japan), goat anti-nectin-2 polyclonal (Santa Cruz Biotechnology, Dallas, TX), rabbit anti-espin1 polyclonal (#PB538, a gift from Dr. Bechara Kachar, NIH) [49 (link)], mouse anti-espin1 monoclonal, mouse anti-N-cadherin monoclonal (BD Bioscience, San Jose, CA), and mouse anti-phospho-myosin light chain2 (Ser19) monoclonal (Cell Signaling, Danvers, MA). Inhibitors used were SMIFH2 (Tocris, Bristol, United Kingdom) and CK-666 (Tocris, Bristol, UK).

Primary antibodies against following proteins were used: Coro1 (ab56820 and ab72212), β-actin (ab20272) from Abcam (Cambridge, UK); Coro3 (K6-444 hybridoma supernatant) [42 (link)]; β3-integrin (HC93 sc-14009) and Gαs (sc-823) from Santa Cruz Biotechnology (Heidelberg, Germany); phosphor-VASP(Ser157) (#3111) from Cell Signaling Technology (Leiden, The Netherlands); GAPDH (6C5-CB1001) from Calbiochem/Merck (Watford, UK); p34-Arc/ARPC2 (07-227) from Millipore/Merck. Secondary antibodies Alexa Fluor 568-conjugated anti-rat or anti-rabbit immunoglobulins (Molecular Probes, Thermo Fisher Scientific, Altrincham, UK) were used for immunofluorescence. IRDye 680 or IRDye 800 anti-mouse and anti-rabbit immunoglobulins (LI-COR Biosciences, Lincoln, NE, USA) were used for Western blot. Human fibrinogen was from Enzyme Research (Swansea, UK), collagen (Kollagenreagens Horm) was from Takeda (Osaka, Japan), and PGI2 was from Cayman Chemical (Ann Arbor, MI, USA). Phosflow Lyse/Fix Buffer and P-selectin were from BD Biosciences (Oxford, UK). Gly-Phe-Hyp-Gly-Glu-Arg (GFOGER) and collagen-related peptide (CRP) were from Cambridge University (Cambridge, UK). U46619 was from Enzo (Exeter, UK). CK666 was from Tocris Bioscience (Abingdon, UK). Thrombin, ADP and FITC, or TRITC-conjugated phalloidin were from Merck (Dorset, UK). Other reagents were from Merck unless otherwise indicated.

Chemical perturbants were acquired from Sigma-Aldrich, except SMHIF2 (Merck), calpeptin (Cytoskeleton), IPA3 and CK666 (Tocris). Perturbants were used at the indicated concentrations. A microsyringe (Hamilton) was used to inject the perturbants into the Petri dish containing the micropillar substrates. Supplementary Table 1 lists the perturbants used, how they were applied (preincubated or added after nuclear envelope breakdown) and a short description of their action.

For chemical perturbations, suspended fibroblasts were pre-incubated with the inhibitors: SMIFH2 (20 μM, Merck Millipore, Billerica, MA), Y27632 (10 μM, Sigma-Aldrich), H1152 (1.6 nM, Merck Millipore), CK666 (200 μM, Tocris Bioscience, Bristol, UK), CK869 (200 μM, Tocris Bioscience, Bristol, UK), C8 inhibitor50 (1 μM, kind gift of William deGrado, UCSF), blebbistatin (20 μM, Sigma-Aldrich), ML-7 (50 μM, Tocris Bioscience, Bristol, UK) and STC (2 μM, Sigma-Aldrich) for 30 min in SCFS media at 37 °C. To inhibit RhoA, fibroblasts were incubated with C3 toxin (2 μg ml^–1, Cytoskeleton, Denver, USA) or Y12 (30 μM, Merck Millipore) for 2 or 3 h, respectively, before the experiments. All reagents were dissolved in dimethylsulphoxide (DMSO) except cell permeable C3 toxin, which was dissolved in 50% (v/v) glycerol. As control we used 0.1% (v/v) DMSO. To block β1-integrins, trypsinised fibroblasts were incubated with α5β1 blocking antibody MAB2575 (Millipore, USA) for 30 min, before the experiments. To block β3-integrins, trypsinised fibroblasts were incubated with 1 μM cilengitide (Selleck Chemicals, Houston, TX, USA) for 30 min, before the experiments. SCFS was conducted in the presence of the respective drug/antibody in the stated concentrations.