Actin polymerization induces MKL1 nuclear accumulation. (A) Untreated (left) and 1-hour TPA-treated (right) HEL-iMKL1 cells were stained for filamentous actin using phalloidin (red) and for DNA using DAPI (blue). Filamentous actin was significantly increased in the large, flattened TPA-treated cells. (B) HEL-iMKL1 cells were treated with TPA for 2 hours and then incubated with the indicated compounds for an additional hour. Left images indicate MKL1 staining; right images overlay MKL1 (green) staining with DAPI (blue) counterstaining. MKL1 accumulated in the nucleus of control (DMSO) cells and cells that had a reduction in monomeric actin (jasplakinolide [iv] and cytochalasin D [iii]), but remained cytoplasmic in cells with increased monomeric actin (latrunculin B [ii]). (C) Graph quantifying the subcellular localization of MKL1 in HEL-iMKL1 cells shown in panel B. No fewer than 75 cells per time point were analyzed from 5 random fields of view in 3 independent experiments. Error bars indicate SEM. All images were taken using a 20× objective.