Figure 6.

CNN3 and CAMK2D Are Targets of CDR1as, which Are Required for Osteogenic Transition

(A) The CNN3 and CAMK2D protein levels in the Hyp with the siCDR1as group were evidently downregulated compared with those in the Hyp with NC group. (B and C) siCDR1as reduced the level of CNN3 (B) and CAMK2D (C) in HPASMC cells induced by Hyp, and this effect was reversed by cotransfecting cells with antagomir. Scale bars, 25 μm. (D) Verification of the effect of knockdown and overexpression on CNN3 and CAMK2D. We used siCAMK2D-3 and siCNN3-1 to knock down CAMK2D and CNN3 in the subsequent experiments. (E) Effects of CNN3 and CAMK2D siRNA and overexpression plasmid transfection on the expression levels of SM22α and BMP2. (F) ALP activity was assayed in siNC-, siCNN3-, siCAMK2D-, VE-, CNN3-, and CAMK2D-overexpressing plasmid-transfected HPASMCs. (G) Quantification of the calcium content in Hyp-induced HPASMCs with or without silencing or overexpression of CNN3 and CAMK2D. ∗p < 0.05, ∗∗p < 0.01 compared with Nor + NC; #p < 0.05, ##p < 0.01 compared with Hyp + NC; &p < 0.05, &&p < 0.01 compared with Nor + VE; $p < 0.05, $$p < 0.01 compared with Hyp + VE. All tests were performed at least three times, and the values are presented as the mean ± SEM.