High MASTL levels promote contractile actin stress fibers. (A) Representative images of F-actin (Phalloidin-Atto) and pMLC (Thr18/Ser19) staining in siControl and siMASTL MDA-MB-231 cells plated on collagen-coated crossbow-shaped micropatterns (37 µm). (B) Quantification of the number of actin stress fibers in A (low, no obvious stress fibers; moderate, one to four stress fibers; high, five or more stress fibers). (C) Analysis of lamellipodium width from A, 38 (siControl) or 59 (siMASTL) cells in total. The width of the lamellipodium was measured from the widest point of the lamellipodium, 38 (siControl) or 60 (siMASTL) cells in total (narrow, 0–1.2 µm; moderate, 1.2–2 µm; and wide, >2 µm; n = 3 biologically independent experiments, Fisher’s exact test). (D) Western blot analysis of phosphorylated (Ser20) and total MLC (pMLC and MLC, respectively) and MASTL protein levels in siControl and siMASTL MDA-MB-231 cells. GAPDH was used as a loading control. (E) Quantification of pMLC relative to total MLC protein levels after MASTL silencing (n = 3 biologically independent experiments, unpaired t test, mean ± SD). (F) Western blot analysis of pMLC (Ser20) and total MLC, MASTL and GAPDH protein levels in siControl, siMASTL, and siMASTL + EGFP-MASTL (24 h MASTL silencing + 24 h expression of siRNA-resistant EGFP-MASTL) MDA-MB-231 cells. Shown is a representative blot of three biologically independent experiments with similar results. See also Table S1.