Actomyosin-mediated forces are required for the formation of tensin3-positive fibrillar adhesions. (A and B) Representative immunofluorescence images of U2OS cells cultured overnight on fibronectin-coated glass stained for (A) tensin3 and vinculin, or (B) tensin3 and fibronectin. (C) U2OS cells were treated in suspension with blebbistatin (50 µM) or an equivalent volume of DMSO, for 60 min. Cells were fixed after spreading on fibronectin-coated glass for 4 h. Note the absence of tensin3- or vinculin-positive structures in blebbistatin-treated cells. (D) U2OS cells cultured overnight on fibronectin-coated glass were treated with blebbistatin (50 µM) or an equivalent volume of DMSO, for 60 min before fixation. Line profiles indicate fluorescences intensity levels of proteins from background subtracted FA images. Note that tensin3 remains at adhesions whilst vinculin disappears after blebbistatin treatment. Data are normalized to control (DMSO-treated) conditions individually for tensin and vinculin. Error bars are SD; n = 5,206 (DMSO); 4,078 (Blebbistatin) adhesions from 45 to 56 cells respectively; data were pooled from three independent experiments; **** indicates P < 0.0001 (Mann Whitney t test). Scale bars in A–C indicate 10 µm.