MASTL regulates GEF-H1, T pm 4.2 , and NM-2B levels and GEFH1 rescues cell spreading, but not cell migration, in MASTL-silenced cells (related to Figure 5 ). (A) Western blot analysis of GEF-H1, Tpm4.2, and MASTL protein levels in siControl, siMASTL#6, and siMASTL#7 MDA-MB-231 cells. (B) Representative GEF-H1 and tubulin staining in siControl, siMASTL#6, and siMASTL#11 MDA-MB-231 cells. Images were acquired on a 3i CSU-W1 spinning disk confocal. (C) Western blot analysis of GEF-H1 and MASTL protein levels in siControl, siMASTL#6, and siMASTL#7 MCF10A cells. (D) Representative Tpm4.2 staining in siControl, siMASTL#6, and siMASTL#11 MCF10A cells. (E) Representative F-actin (Phalloidin-Atto) and DAPI staining in siControl and siMASTL#11 MDA-MB-231 cells transfected with EGFP or EGFP-GEF-H1 for 24 h and plated on collagen for 2 h. Images were acquired on a 3i CSU-W1 spinning disk confocal. (F) Migration track length of siControl and siMASTL#11 MDA-MB-231 cells transfected with EGFP or EGFP-GEF-H1 for 24 h. Cells were imaged every 10 min for 600 min in total (n = 3 biologically independent experiments, mean ± SD, unpaired t test). See also Table S1.