MASTL regulation of vinculin-positive focal adhesions and SRF activity (related to Fig. 8 ). (A) Quantification of paxillin levels after MASTL silencing. Protein levels are normalized by GAPDH (n = 3 biologically independent experiments, mean ± SD, unpaired t test). (B) Representative images of F-actin (Phalloidin-Atto), vinculin, and DAPI staining in siControl and siMASTL (48 h silencing) MCF10A cells plated on collagen for 2 h, and quantification of focal adhesion size based on vinculin staining (three independent experiments). Images were acquired on a 3i CSU-W1 spinning disk confocal. (C) Representative staining of vinculin and E-cadherin in confluent siControl, siMASTL#6 and siMASTL#11 MCF10A cells. Images were acquired on a 3i CSU-W1 spinning disk confocal. (D) Relative SRF luciferase activity. MCF7 cells expressed EGFP control and EGFP-MASTL E167D together with the SRF reporter 3D.Aluc and RLTK Renilla luciferase transfection control under serum-starved conditions and after stimulation with 20% serum. (E) Western blot of β-actin in F- and G-actin fractions of MCF10A cells treated with DMSO or jasplakinolide (Jasp.; 0.1 µM, 30 min) or silenced with siControl, siMASTL#6, or siMASTL#7 (48 h). (F) Quantification of F-actin protein levels relative to G-actin in MCF10A cells after treatments from B (n = 3 biologically independent experiments, mean ± SD, unpaired t test). See also Table S1.