Figure 5. Functional analyses of THSD1 in HUVECs.

A) Transfection of HUVECs with targeting siRNA inhibited THSD1 relative to control siRNA-treated cells as determined by Western. The THSD1 open reading frame in pCR3.1 was engineered by silent mutagenesis to be siRNA-resistant and was used as a template for both WT and mutant THSD1 constructs. Co-transfection of THSD1 siRNA and the vector expressing wild-type THSD1 restored expression. In contrast, the L5F mutant resulted in undetectable protein expression, while R450X led to a truncated protein. C-terminal (C-ter) and N-terminal (N-ter)-targeting THSD1 antibodies. B) THSD1 and talin co-immunoprecipitate each other in HUVEC cells. Cell lysates of HUVECs transfected with WT THSD1 vector were subjected to immunoprecipitation (IP) with either an anti-C-ter THSD1 antibody (left panel) or anti-talin antibody (right panel) and immunoblotted with anti-talin, anti-C-ter THSD1 or anti-GAPDH antibody. C) Cell adhesion defects to collagen I in THSD1 siRNA-treated cells are rescued by co-transfection of siRNA-resistant wild-type but not variant THSD1 from IA-patients. * and ** denotes P<0.05 and P<0.001, respectively. D) (top) light microscopy imaging and immunofluorescence analyses of cells treated with a control or THSD1 siRNA; (bottom) quantification for cell areas (left) and focal adhesion numbers (right). Cell peripheries are shown in blue. In immunofluorescence staining, cells were stained with anti-C-ter THSD1, anti-paxillin, anti-pFAK (Y397) and anti-vinculin antibodies. A significant reduction in cell size and focal adhesion number was observed in THSD1 siRNA-treated cells.