Spry1 and Spry4 differently regulate Akt activation, hAoSMC differentiation and proliferation.

<p>A) Representative immunoblot shows that overexpression of Spry1 increases VSMC marker CNN1, ACTA2 and SM22α expression, whereas overexpression of Spry4 decreases expression of these markers. B) Quantification of VSMC marker protein expression from at least three independent immunoblotting experiments show that overexpression of Spry1 significantly increases ACTA2, CNN1 and SM22α, whereas overexpression of Spry4 has small but statistically significant decrease of CNN1 and SM22α. C) Quantification of RT-qPCR from three independent experiments shows that overexpression of Spry1 increases VSMC marker gene mRNA (<i>SM22α</i>, and <i>CNN1</i>), whereas over expression of Spry4 decreases these gene mRNA expression. D) Representative immunoblot to show that Spry1 has little effect on pERK but increases pAkt, while Spry4 decreases both pERK and pAkt in hAoSMC. E) Quantification of the relative levels of pERK vs. ERK and pAkt vs. Akt from at least three independent immunoblotting experiments. F) Flow cytometric analysis shows that Spry1 increases, while Spry4 decreases hAoSMC proliferation as indicated by the percentage of Ki67 positive cells. Quantification is from three independent experiments. G) Representative immunoblot shows that Spry1 increases cyclinD1 expression. H) Cell migration wound healing assay. Representative images show that more Myc-positive Spry1 overexpressing cells migrated into wound area compared to Myc-positive Spry4 overexpressing cells 48 hours after wounding. Mitomycin treatment was used to eliminate the effect of cell proliferation. Note: *: p<0.05; **: p<0.01.