Figure 1

(A) Immunofluorescence (IF) staining of integrin α5 (ITGA5) and β1 (ITGB1) in MGC803 cell line overexpressing TSPAN4-GFP. Scale bar, 10 μm. (B) IF staining of active integrin β1 (HUTS-4 or 12G10) in TSPAN4-GFP-overexpressing MGC803 cells. Scale bar, 10 μm. (C) Live-cell images of NRK cells transfected with TSPAN4-GFP and integrin α5-mCherry. Scale bar, 10 μm. (D) NRK cells were co-transfected with TSPAN4-mCherry and integrin α5-GFP, and time-lapse images were taken every 8 min. Scale bar, 1 μm. (E) 3D reconstitution of TSPAN4-mCherry and integrin α5-GFP on migrasomes and retraction fibers. T, top view; L, lateral view; B, bottom view. Scale bar, 2 μm. (F) TSPAN4-GFP-expressing NRK cells were cultured on different ECM proteins (fibronectin, laminin 511 and collagen I), and the number of migrasomes formed on each was counted. (G) The expression level of different integrin α subunits in TSPAN4-GFP-expressing NRK cells was analyzed by qPCR. (H) TSPAN4-GFP-expressing NRK cells were transfected with control or ITGA5-targeting siRNA for more than 24 h, then plated on the FN-, LN511- or Col I-coated chamber overnight, and confocal pictures regarding migrasome number for each cell were taken. The western blotting analysis (the right panel) showed the knocking down efficiency. (I) NRK cells expressing TSPAN4-GFP alone (control) or TSPAN4-GFP with mCherry-tagged integrin α3 were cultured on different ECM proteins and the number of migrasomes per cell was counted. Data were analyzed with two-tailed t-tests (GraphPad Prism 5); ^***P < 0.001. (J) CHO cells were transfected with pEGFP-N1 (control) or integrin α1-GFP and seeded into chambers coated with different ECM proteins, and the number of migrasomes per cell was counted. Data were analyzed with two-tailed t-tests (GraphPad Prism 5); ^***P < 0.001.