Durotactic substrates altered the responses of U251 cells to inhibition of NMMII and ROCK Y27632, responsible for cytoskeleton assembly and cell contractility. U251 cell morphology subsequent inhibition was sensitive to the durotactic micropatterned substrates compared to uniform substrates. Effects of blebbistatin (a) and ROCK Y27632 (b) on cellular spread area, elongation (Feret diameter), A.R. and Circularity for each substrate stiffness. Treated cells (bars) are compared with that in control cells (dotted lines). Relative to the morphologic cell response on uniform substrates, U251 decreased their spread area and elongation on the durotactic gradient substrates. Cell migration observed by time-lapse video microscopy was similar across all mechanically uniform substrates evaluated. Cell track positions were used to calculate the displacement (c), speed (d) of migration and the mean squared displacement (e,f) with blebbistatin (25 µM, 8 h), and ROCK Y27632 (10 µM, 8 h). Statistical significance indicated by * for p < 0.05, and *** for p < 0.0001, assessed by Tukey one-way ANOVA test. Error bars indicate S.E.M.