Figure 3. Notch1 was the direct target of miR-34b and involved in regulating VSMCs calcification. (A) Schematic of the miR-34b putative target sites in mouse Notch1 3′-UTR and alignment of miR-34b with WT and MUT Notch1 3′-UTR, showing pairing. The mutated nucleotides were underlined. (B) Western blot analysis showed the level of Notch1 protein in VSMCs treated with 3.5 mM of Pi. (C) VSMCs were transfected with scramble miRNA mimics, inhibitor or miR-34b mimics, miR-34b inhibitor, respectively, and harvested for the examination of Notch1 protein by western blot analysis. (D) The WT-Notch1 3′-UTR and the MUT- Notch1 3′-UTR reporters were co-transfected with miR-34b mimics or control oligos into VSMCs. Forty-eight hours after transfection, the luciferase activities were measured. (E) Notch1 was knocked down successfully by Notch1 siRNA. (F) The expression of miR-34b was assessed by qRT-PCR in VSMCs induced by 3.5 mM of Pi after transfecting with scramble siRNA or Notch1 siRNA. (G–I) ALP activity, OC secretion and Runx2 expression were determined in VSMCs induced by 3.5 mM of Pi after transfecting with scramble siRNA or Notch1 siRNA. n = 3. The data were expressed as mean ± SD, *p < 0.05; **p < 0.005.