Effect of Rho, ROCK, MLC signaling inhibition on the invasiveness and morphology of PR9692 cells. (A) Immunodetection of recombinant dnRhoA, dnMLC and NPTII proteins in PR9692 cells. (B) 3D in vitro collagen invasion. Treatment of PR9692 cells with metalloproteinase inhibitor GM6001 does not reduce the cell invasion. Inhibition of ROCK by Y-27632 and inhibition of non-muscle myosin II ATPases activity by Blebbistatin in PR9692 cells as well as inhibition of both RhoA in PR9692-dnRhoA and MLC in PR9692-dnMLC lead to decreased ability of these cell lines to invade 3D collagen. (C) Morphology assay in 3D collagen in vitro. Activity of RhoA is required for the rounded morphology of the PR9692 cell line. Inhibition of ROCK by Y-27632 and inhibition of RhoA in PR9692-dnRhoA lead to the amoeboid-mesenchymal transition in 3D collagen. Inhibition of metalloproteinases by GM6001, inhibition of non-muscle myosin II ATPases activity by Blebbistatin in PR9692 cells and inhibition of MLC activity in PR9692-dnMLC cells have no significant effect on morphology.