Distribution of LL5s in MCF-10A spheroids and its dependency on laminin receptor integrins. (A) Schematic diagram of the overlay method for three-dimensional culture of MCF-10A cells in Matrigel (Debnath et al., 2003). MCF-10Aeco cells seeded onto a solid bed as a single-cell suspension proliferate and start to form clusters (spheroids) in three-dimensional culture. (B) Representative confocal microscopic images of MCF-10Aeco spheroids. Spheroids at day 3 were fixed and immunostained with antibodies against LL5s and laminin-5. Nuclei were stained with Hoechst 33342. The outer surface of the spheroids covered with laminin-5–positive BM is the basal side of the cells. (C and D) Confocal images of MCF-10Aeco spheroids treated with the indicated siRNAs and stained for LL5s and integrins. Integrin αv is known to localize to FAs. Note that knockdown of integrin β1 + β4 is estimated by the integrin α6 signals, owing to the limited availability of primary antibodies from different host species for simultaneous staining. The reduction of integrin α6 after integrin β1 + β4 knockdown was confirmed in independent experiments (Fig. S4). In integrin α3 knockdown cells, integrin α6β4 is also partially reduced for unknown reasons, but LL5s are still retained at the basal cortex. (B–D) Insets are magnified images of the boxed areas. (E–G) Quantitative analyses of the fluorescent signals at the basal cortex in the spheroids. For each condition, >35 cells were analyzed. The data were transformed into a relative fluorescence index, and the values of the mock control were set as 1. LL5 localization is inhibited when integrins α3 + α6 or β1 + β4 are knocked down. Note that integrin αv is unaffected by knockdown of integrins α3 + α6 or β1 + β4, whereas the signals for LL5s are abolished. The results are presented as means ± SEM (*, P < 0.01 vs. the mock control). KD, protein that was knocked down. Bars, 10 µm.