Subcellular localization of LL5s in MCF-10A cells. (A) Selected images from a time-lapse video of MCF-10Aeco cells expressing RFP-LL5α and GFP (Video 1). GFP allows visualization of the cell shape. The images were collected soon after cell seeding. Accumulation of RFP-LL5α (arrowheads) is initiated upon cell attachment to the substratum. (B) Comparison of the distributions of LL5β and FAs visualized with antivinculin antibodies. Representative images of cells stained for LL5β and vinculin at 2 (top), 8 (middle), and 10 (bottom) h after seeding are shown. Insets are magnified images of the boxed areas. The percentage of colocalized pixels and the Pearson’s correlation coefficients (R(r)) between vinculin (G) and LL5β (R) channels are presented in the figures. (C) Comparison of the distributions of RFP-LL5α and cell–substratum attachment sites visualized by interference reflection microscopy (grayscale). (D and E) Colocalization of LL5s with laminin-5 deposition and laminin receptor integrins. (D) Autocrine laminin-5 is secreted and deposited in a flower pattern on the glass surface by the cells. (E) The colocalization of fluorophores in D (a–c) was quantified inside the whole cell area and the LL5-concentrated area. Colocalization analyses are similar to Fig. 1 E. Bars: (A and B) 10 µm; (C and D) 5 µm.