Ck 869

α-sarcin was purchased from Santa Cruz. ³⁵S methionine and ³⁵S cysteine were purchased from Perkin Elmer. Alexa FluorA647 succinimidyl ester was purchased from ThermoFisher Scientific. Inhibitors were purchased from commercial vendors as follows: Aim-100 (Apexbio), Wiskostatin (Sigma), Dynasore (Sigma), Pitstop-2 (Sigma), CK-869 (Sigma), Pirl1 (Hit2leads). Anti-EMCV mouse polyclonal antibodies were provided by Michael Diamond. Anti-poliovirus antibodies were provided by Nihal Altan-Bonnet. Other antibodies were obtained from commercial vendors as follows: Anti-coxsackie virus B3 antibodies (ThermoFisher), Anti-adenovirus A5 antibodies (ThermoFisher), Anti-influenza A virus NP antibodies (Millipore), Anti-TNK2 (A11) (Santa Cruz), Anti-WASL (Abcam and Sigma), Anti-actin, clone C4 (Sigma), Anti-NCK1 (Millipore), enterovirus D68 Ab (GeneTex), Anti-HA (ThermoFisher), Anti-Flag (GenScript), Anti-c-Myc (Invitrogen), Anti-double stranded J2 antibodies (Scicons).

EBV-transformed B cells (JY line) were cultured in RPMI 1640 GlutaMax, supplemented with 10% Fetal Calf Serum, minimum essential amino acids, HEPES, sodium pyruvate and Streptomycin/penicillin solution (Invitrogen) Cells were routinely screened for mycoplasm contamination using the MycoAlert mycoplasma detection kit (Lonza). For the stable expression of GFP, LifeAct-GFP, LifeAct-RFP, 1 × 10⁵ JY cells were transduced with a lentiviral vector encoding LifeAct-TagGFP2 or LifeAct-TagRFP (Ibidi) over 6 h in RPMI-1640 containing 5% FCS and polybrene (4 μg ml⁻¹). Transfected cells were selected by culturing them in the presence of 2 μg ml⁻¹ puromycin for 7 days, added 24 h after transfection. Bright GFP-positive cells were further selected by flow cytometry sorting. Where indicated, cells were treated with Y27632 (Abcam) at 10 μM, CK 869 (Sigma-Aldrich) at 50 μM or the equivalent concentration of DMSO (10⁻³). No treatment toxicity was detected in a window of 16 h. Ibitreat^TM (Ibidi) plastic plates were coated with 1,5 μg ml⁻¹ collagen type IV from human placenta or 10 μg ml⁻¹ fibronectin from human plasma (both from Sigma-Aldrich) diluted on PBS. Matrices were incubated ON at 4 °C and 1 h at 37 °C before washing with medium and cell seeding.

The following chemicals were used at the indicated concentrations: ionomycin (Iono, 2 μM), phorbol 12-myristate 13-acetate (PMA, 162 nM), CK-869 (100 μM), KN-93 (0.25 μM), KN-62 (2.5 μM), STO-609 (5 μM), and aphidicolin (15 µM) were all obtained from Sigma-Aldrich, whereas cyclosporine A (1 µM) and 187-1 (NWASPi, 3 µM) were obtained from TOCRIS Bioscience. Of note, aphidicolin and NWASPi are very unstable and lose activity within 7 d and 2 d, respectively, post reconstitution and storage at –20°.

Cells were treated with 50 µM blebbistatin (Sigma-Aldrich, St Louis, MO USA) to inhibit myosin II ATPase activity, 400 nM latrunculin A (Enzo Life Sciences) to sequester actin monomers, 1 µM thapsigargin to inhibit Ca²⁺ uptake into the ER, 1 µM cytochalasin D (Sigma) to depolymerize actin, 10–50 µM cyclosporin A (Sigma) to inhibit calcineurin, 1–10 µM KN93 or KN62 (Tocris) to inhibit Ca²⁺/calmodulin-dependent protein kinase II, 10–20 µM Y27632 (Sigma) to inhibit ROCK, 2.5 µg/µl C3 transferase (Cytoskeleton) to inhibit Rho1, 1 µM nocodazole (Sigma) to depolymerize microtubules, 10–20 µM SMIFH2 (Sigma) to inhibit formins and 50–100 µM CK666 or CK869 (Sigma) to inhibit the ARP2/3 complex.