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. 2019 Dec 20;21(1):76. doi: 10.3390/ijms21010076

Figure 5.

Figure 5

Figure 5

MiR-204 antagomir promotes aortic valve calcification through up-regulation of Runx2 and Osx expression. (A) Isolated mouse aortic valves were treated with irrelevant miR (control) or miR-204 antagomir for three days. Representative immunofluorescence images of 5 separate experiments show that miR-204 antagomir increases the levels of Runx2 and Osx. Runx2 and Osx are showing in red. The nuclei are showing in blue. Glycoproteins are showing in green to outline tissue and cells. Original magnification 40× objective. (B) Isolated mouse aortic valves were untreated or treated with irrelevant miR or miR-204 antagomir. Additional valves were treated with miR-204 antagomir in the presence or absence of siRNA (100 nM) specific to Runx2 or Osx. Then, all valves were incubated in the conditioning medium for 6 days. Representative images of Alizarin Red S staining and spectrophotometric analysis of Alizarin Red S stains show that miR-204 antagomir augmented the formation of calcium deposits (brick red and black) in mouse aortic valves. Knockdown of Runx2 or Osx attenuated calcium deposit formation caused by antagonizing miR-204. Mean ± SE; n = 5; * p < 0.05 vs. untreated (conditioning medium alone) or miR-C (irrelevant miR+conditioning medium); # p < 0.05 vs. miR-204 antagomir; ≠ p < 0.05 vs. miR-204 antagomir+control siRNA. Scale bar = 200 μm.