Myosin IIa, but not myosin IIb, is required for the peripheral distribution of Rab27a-positive WPBs. (Ai) Immunostaining of paxillin (black) in HUVECs expressing scrambled (SCR) and shRNA targeting myosin IIa (shMyoIIa). (Aii) Spinning disk confocal microscopy of the height map (depth was defined as the distance of WPBs to the basal membrane) of WPBs relative to the basal membrane in these HUVECs. Height of the basal membrane was defined as 0 μm (red), and the apical membrane was indicated in blue. (B) Statistics for the height of WPBs as in A. The height of WPBs was evenly divided into 3 layers: basal (red), middle (green), and apical (blue). (C) Representative images of immunostaining of VWF (yellow), F-actin (cyan), and paxillin (magenta) in cells expressing SCR and shMyoIIa. Dashed lines indicated FA-anchored SFs. (D) Percentage of WPBs along stress fibers and length of FA-anchored SFs, as in D. **P < .01, ***P < .001. (E) Representative real-time images and height maps with blebbistatin treatment in cells expressing P-selectin-lum-mCherry (PSL-lum-mCherry) and Lifeact-GFP. (F) Representative images of immunostaining of VWF (yellow), f-actin (cyan), and Rab27a (magenta) in cells expressing SCR and shMyoIIa. The magnified panels showed the peripheral region (a) and the peri-nuclear region (b). The peripheral region was defined as the region whose distance to nuclear was more than 10 μm. Arrows indicate the Rab27a-positive WPBs and arrowheads indicate the Rab27a-negative WPBs. (G) Quantitative analysis of the number of Rab27a-positive WPBs in perinuclear and peripheral regions (mean ± standard deviation. ***P < .001, Student’s t-test (scale bars, 5 μm; mean ± standard deviation).