Src activation induces Septin2 localization to podosomes in ECs. (A) Confocal images showing localization of endogenous Septin2 to Src-induced functional endothelial podosome rosette (indicated by white boxes). HPAECs expressing RapR-Src were seeded onto an Alexa Fluor 405–conjugated gelatin (Alexa405-gelatin). RapR-Src was activated for 1 h. Cells were fixed and stained for F-actin and endogenous Septin2. Functional podosomes are defined as sites of dense F-actin signal colocalizing with areas where the fluorescent gelatin signal is quenched (scale bar = 10 µm). (B and D) Airyscan images depicting Septin2 colocalizing with known podosome markers MT1-MMP (B; scale bar = 5 µm) and Tks5 (D; zoomed images showing areas outlined with white rectangles; scale bar = 3 µm). Cells were prepared as in A, seeded onto unlabeled gelatin substrate, and fixed and stained after 0.5 h of RapR-Src activation. (CandE) Imaris image analysis software was used to create a 3D surface rendering of endogenous Septin2 colocalizing with MT1-MMP (C) and Tks5 (E) in endothelial podosomes.