Spry1 is essential to maintain hAoSMC marker gene expression.

<p>A) Co-immunofluorescence staining of Spry1, Spry4 and ACTA2 on mouse artery sections show that both Spry1 and Spry4 are expressed in normal arterial smooth muscle cells. Images are from original 400× magnification. B) Immunohistochemistry of Spry1 and Spry4 on human atherosclerotic artery samples showed the expression of Spry1 and Spry4 in artery media smooth muscle cells, and a decreased expression of Spry1 and Spry4 in neointima cells. Images are from original 200× magnification. C) Quantification of RT-qPCR from three independent experiments shows efficacy of knockdown (kd) of Spry1 and Spry4 mRNA by their respective shRNAs compared to a non-targeting sequence (NT). D) Quantification of RT-qPCR from three independent experiments shows that knockdown of Spry1 decreases, whereas knockdown of Spry4 increase VSMC differentiation gene expression at the mRNA level. E) Representative immunoblot showing that knockdown of Spry1 results in a decrease in total Akt and ERK proteins levels, as well as pAkt and pERK. The expression of VSMC marker genes SMTN-B and SM22α is decreased by knockdown of Spry1, whereas knockdown of Spry4 increases pAkt and pERK levels as well as SMTN-B and SM22α. F) Quantification of VSMC marker protein levels from at least three independent immunoblotting experiments. G) Immunofluorescence staining shows down-regulation of SM22α following knockdown of Spry1. Images are from original 400× magnification. H) Quantification of phospho-histone 3 (pH3) immunofluorescence staining from triplicate experiments shows that knockdown of Spry1 reduces pH3 staining, while knockdown of Spry4 increases pH3 staining. Note: a: adventia, m: media, i: intima l: lumen; **: p<0.01, *: p<0.05.