MYO18B is required for stress fiber assembly and FA maturation. (A) Representative wide-field epifluorescent images of HeLa cells stained with rhodamine-conjugated phalloidin. Accumulation of actin filaments in the cytosol was observed in MYO18B-KO cells. Bar, 10 µm in the original image and 2 µm in the magnified image. See also Fig. S2 A and Video 2. (B) Representative CLSM images of WT and MYO18B-KO HeLa cells stained with vinculin. Bar, 10 µm. (C) Quantitative results of vinculin puncta >0.5 µm² in WT and MYO18B-KO HeLa cells (mean ± SD; n = 15 images; N = 3 biological replicates; unpaired two-tailed t test; **P ≤ 0.01). See also Fig. S2, B and C. (D) Representative live imaging of WT and MYO18B-KO U2OS cells transiently expressing paxillin-mCherry (CLSM). An example frame of a 9 h time-lapse image session is shown on the left, and the lifetime of one FA (black arrow) is shown in a time series on the right. Images are inverted for a better contrast. Bar, 10 µm in original images (left) and 2 µm in magnified montage (right). See also Video 3. (E) Fitted RFI curve of FAs, as indicated in Fig. 4 D. The maximum intensity is set as unity. The black arrow indicates focal adhesion lifetime which is the duration between half maximum of the fitted curve. See STAR methods for the method details of curve fitting. (F) Quantitative results of the FA lifetime in WT and MYO18B-KO U2OS cells (n = 5 FAs; mean ± SD; unpaired two-tailed t test; ***P ≤ 0.001).