Megakaryocyte differentiation of HEL cells promotes MKL1 nuclear accumulation. (A) Immunofluorescence images of MKL1 localization in HEL-iMKL1 cells. On the left is MKL staining alone; right, overlay with DAPI and phalloidin staining. N indicates nuclear; C/N, pancellular; and C, cytoplasmic. (B) Doxycycline-treated HEL-iMKL1 cells were serum starved overnight and treated with TPA for the indicated times. The percentage of cells with exclusively nuclear localization (black bars) increases during the first 3 hours after TPA treatment and then declines. No fewer than 75 cells per time point were analyzed from 5 random fields of view in 3 independent experiments. Error bars indicate SEM. (C) Western blot analysis of HEL-iMKL1 cells after treatment with TPA for 3 hours. Cells were fractionated into nuclear and cytoplasmic compartments and cell lysates were probed for MKL1, TATA-binding protein (TPB; nuclear), and GAPDH (cytoplasmic) expression. Note that the nuclear fraction lacks cytoplasmic contamination.