MASTL supports cell migration without influencing focal adhesion dynamics. (A) Representative phase contrast images from a 90-min time-lapse movie of siControl and siMASTL MDA-MB-231 cells (Nikon Eclipse Ti-E widefield microscope, Hamamatsu Orca C13440 Flash 4.0 ERG [b/w] sCMOS camera and Plan Apo lambda 20×/0.80, WD 1,000-µm objective). (B) Migration maps based on X and Y coordinates of siControl and siMASTL cells, recorded every 10 min, 700 min in total (9 tracks per condition shown with different colors; Nikon Eclipse Ti-E widefield microscope). (C) Migration track length of siControl and siMASTL cells (45 cells/condition in total, n = 3 biologically independent experiments, mean ± SD, unpaired t test with Welch’s correction). (D–G) Quantitative image analysis of focal adhesion dynamics based on live-cell imaging of endogenously tagged mEmerald-Paxillin in MDA-MB-231 cells. Visualization of the entire set of focal adhesions recorded every minute for a total of 40 min (D). Quantification of focal adhesion lifetime (E), focal adhesion assembly rate (F), and focal adhesion disassembly rate (G). Total number of cells/condition is indicated in the figure and Table S1. (H) Western blot analysis of MASTL, phospho-paxillin (Tyr118), and paxillin levels in siControl and siMASTL (48 h silencing) MDA-MB-231 cells. GAPDH used as a loading control. (I) Quantification of phospho-paxillin relative to paxillin after MASTL silencing (n = 3 biologically independent experiments, unpaired t test, mean ± SD). See also Table S1.