MLCK mediates substrate stiffness-dependent neutrophil transmigration. (A) TNF-α–activated HUVEC monolayers were pretreated with appropriate control (DMSO or IgG antibody), cytoB, or VE-cadherin antibody (VEcad Ab) for one hour. Neutrophils were plated onto the HUVECs, and the fraction of transmigration was quantified on soft (0.87 kPa) and stiff (280 kPa) substrates. (B) TNF-α–activated HUVEC monolayers were pretreated with DMSO, blebbistatin, or ML-7. Neutrophils were plated onto the HUVECs, and the fraction of transmigration was quantified on soft (0.87 kPa), intermediate (5 kPa), and stiff (280 kPa) substrates. Also shown is the fraction of transmigration for ML-7–treated neutrophils through TNF-α–treated monolayers. (A-B) Bars represent average fraction of transmigrated cells. Error bars represent SE from at least 3 independent experiments. (A) *P < .05 with IgG antibody control using Student t test. (B) *P < .05, **P < .01 between treated monolayers and DMSO control using Student t test. (C) Schematic illustrating a possible mechanism for how pretreatment of HUVEC monolayers with ML-7 normalizes the effects of substrate stiffness in neutrophil transmigration. Before ML-7 treatment (left), neutrophil adherence to the endothelium induces a signaling cascade, which activates MLCK and results in endothelial cell contraction (black arrows) and gap formation. Because the cells can presumably exert more traction on a stiffer substrate, they are capable of creating larger gaps on the stiff substrate, ultimately allowing more neutrophils to transmigrate through. Treatment of the endothelium with ML-7 causes inhibition of contraction on the stiff substrate. The soft substrate is unaffected, possibly because contraction was already suppressed to some degree, and ML-7 treatment did not produce further effects.