Fig. 4

25HC activates αvβ3 integrin in epithelial cell focal adhesions and macrophage podosomes. a Human lung epithelial cells (A549 cells) or differentiated THP-1 macrophages were subjected to various treatments and immunostained as indicated. In untreated (UT) A549 and THP-1 cells (first row), αvβ3 integrin, immunostained using the antibody LM609, was present in paxillin-positive focal adhesions and podosomes, respectively. In contrast, active αvβ3 integrin staining, indicated using the antibody AP5 (second and third row), was found almost exclusively in cells treated for 2 h with 0.5 µM 25HC compared with DMSO-treated controls. Merged images show the co-localization of αvβ3 integrin (total or activated) with focal adhesions/podosomes. Bar, 10 µm. b Quantification of the number of focal adhesions (FAs) and AP5⁺ FAs in A549 cells (three experiments, >12 cells per experiment; n ≥ 30). c Quantification of the number of podosomes and AP5⁺ podosomes in THP-1 cells (three experiments, 25 cells per experiment; n ≥ 70). Two- and One-tailed Wilcoxon rank-sum tests were performed to evaluate differences between total and AP5⁺ FA/podosome counts, respectively. Graphs depict mean ± SEM. *p ≤ 0.05