Igg nonspecific antibody

Inhibitors in the 3D cell culture were used at the following concentrations: 10 μM Y-27632 to inhibit ROCK (Abcam); 50 μM blebbistatin (Abcam) to inhibit myosin II; and 1 μg ml⁻¹ monoclonal integrin-β1-blocking antibody (Abcam, P5D2). Vehicle-alone controls for these inhibitors were as follows: DMSO for blebbistatin, and latrunculin-a; deionized water for Y-27632; and IgG nonspecific antibody (Sigma-Aldrich, I5381) for integrin-β1-blocking antibody. Y-27632 and blebbistatin were added to the culture medium directly. Integrin β1-blocking antibody was incubated with Huh7 cells on ice for 1 h before encapsulation in IPNs and added to the culture medium directly.

Inhibitors were implemented in invasive morphology and/or migration assays at concentrations as follows: 10 μM GM6001 (Millipore) or 100 μM marimastat (Tocris) to broadly inhibit MMPs; 70 μM NSC23766 to inhibit Rac1 (Tocris); 10 μM Y-27632 to inhibit ROCK (Sigma); 100 μM CK-666 to inhibit Arp 2/3 (Sigma); 1 μg mL⁻¹ monoclonal β1 integrin-blocking antibody (Abcam, P5D2); 50 μM Blebbistatin (Abcam); and 2.5 μM Latrunculin-a (Tocris). Vehicle-alone controls for these inhibitors were as follows: DMSO for GM6001, marimastat, CK-666, Blebbistatin, and Latrunculin-a; deionized water for NSC23766 and Y-27632; and IgG nonspecific antibody (Sigma, I5381) for β1 integrin-blocking antibody. All inhibitor concentrations followed those used in similar studies: GM6001^{6 (link),47 (link)}, marimastat^{48 (link)}, NSC23766^{15 (link)}, Y-27632^{42 (link)}, CK-666^{49 (link)}, β1 integrin-blocking antibody^{42 (link)}, Blebbistatin^{31 (link),50 (link)}, and Latrunculin-a^{50 (link)}. Drug concentrations were also verified in-house using gelatin degradation assays and traction force microscopy experiments (described elsewhere in Methods).