Fig. 2. Loss of Pfn1 expression alters cell-matrix and cell-cell adhesions in HUVECs: (A) Vinculin-immunostaining shows a dramatic reduction in FA formation when Pfn1 expression is silenced (C, control siRNA; P, Pfn1-siRNA). Bar, 20 µm. (B) A bar graph shows a significantly (P<0.001) higher FA density (number of FA/100 µm² of cell area) in control cells (6.1±2.1) than in Pfn1-deficient cells (2.7±1.9). These data are based on analyses of 56 control and 69 Pfn1-deficient cells that were randomly selected from two independent experiments. (C,D) VE-cadherin and ZO-1 immunostaining show that VEGF stimulation causes loss of junctional staining of these proteins (arrow) and creation of paracellular holes (arrowhead) in control cells. VEGF-induced delocalization of VE-cadherin and ZO-1 from cell-cell junctions is significantly inhibited in Pfn1-deficient ECs. Bar, 20 µm. (E) Immunoblot data show no appreciable change in the expression of VE-cadherin and ZO-1 between control and Pfn1-siRNA treated cells (the GAPDH blot serves as the loading controls).