MASTL associates with MRTF-A and regulates nuclear retention of MRTF-A. (A) Live-cell imaging of siControl and siMASTL MCF10A cells stably expressing pIND20-MRTF-A-GFP. Cells were serum starved (24 h) before imaging and MRTF-A-GFP translocation was followed after adding 20% serum (LSM 800 confocal microscope; Zeiss). Analysis of MRTF-A nuclear intensity in siControl and siMASTL cells 0, 120, 240, and 600 s after serum stimulation based on live-cell imaging of 8–9 cells/condition is shown (n = 3 independent experiments). (B) Nuclear/cytoplasmic ratio of MRTF-A in EGFP control, EGFP-MASTL WT, EGFP-MASTL G44S, and EGFP MASTL E167D–overexpressing MCF7 cells fixed 0, 2, or 5 min after adding serum and stained for MRTF-A (n = 3 independent experiments). (C) Western blot of MRTF-A and MASTL in GFP immunoprecipitations and original lysate in EGFP control, EGFP-MASTL WT, and EGFP-MASTL G44S–overexpressing MCF7 cells (n = 3 independent experiments). (D-F) FLIP representative images (D) and analysis (E and F) of MRTF-A nuclear translocation through repeated bleaching of the cytoplasmic MRTF-A-GFP pool in siControl and siMASTL MCF7 cells after 10 min of serum stimulation. Representative fluorescence intensity curves from two individual cells (E) and quantification of rate of decay (F) are shown (n = 3 biological replicates, with 13 cells in total/condition). See also Table S1.