MKL1-deficient dHL-60 cells show impaired uropod retraction associated with downregulation of MYL9. (A) Knockdown of MKL1 induces a uropod retraction defect. dHL-60 cells were left to adhere to fibronectin-coated glass coverslips for 20 minutes, washed, then uniformly stimulated with fMLP for 2 minutes and fixed in 4% paraformaldehyde. Cells were then stained for F-actin and phosphorylation of MLC (pMLC). The dHL-60 MKL1 F-actin staining intensity was increased threefold in the merged (overlay) image. Images were taken with an ×40 lens. White arrow indicates extended uropod. (B) Quantification of the frequency of uropod extension in dHL-60 MKL1 cells observed in (A). More than 250 cells were analyzed in each condition in 2 independent experiments. (C) Inhibition of myosin II interaction with F-actin using blebbistatin (100 µM) induced a uropod retraction defect in dHL-60 WT cells. Images were taken with a ×63 lens. Scale bars = 10 μm. (D) mRNA expression in HL-60 SCR and MKL1 cells analyzed by qRT-PCR. (E-F) Myosin light chain expression was evaluated by western blot. ***P < .001.