LL5s regulate α3 integrin localization in MCF-10A spheroids. (A and B) LL5s stimulate recruitment of integrin α3 to the basal cell cortex. (A) Confocal images of MCF-10Aeco spheroids treated with the indicated siRNAs and stained for LL5s, myc tag, and integrin α3 (pAb) are shown. Insets are magnified images of the boxed areas. (B) The fluorescent signals at the basal cortex were analyzed and plotted. For each condition, >137 cells were analyzed. The fluorescence intensity of the mock control was set as 1. (C and D) Knockdown of either LL5α or -β reduces the localization of integrin α3 at the basal cortex in MCF-10Aeco spheroids. For each condition, >60 cells were analyzed. To detect integrin α3, two different antibodies recognizing different regions of the integrin (a pAb against the cytoplasmic region and an mAb [P1B5] against the extracellular domain) were used in consideration of possible epitope masking by conformational changes or binding to other molecules. The changes in the integrin α3 signals are the same for the pAb and mAb. (B–D) The results are presented as means ± SEM (*, P < 0.01 vs. the mock control). KD, protein that was knocked down. Bars, 10 µm.