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. 2012 Apr 13;7(4):e33126. doi: 10.1371/journal.pone.0033126

Figure 1. Effects of ghrelin on alkaline phosphatase (ALP) activity, Runx2 protein expression, bone morphogenetic protein-2 (BMP-2) mRNA expression and calcium deposition in calcifying vascular smooth muscle cells (CVSMCs).

Figure 1

(A) Effect of ghrelin on ALP activity. The cells were cultured with or without 10−6 mol/L ghrelin for the indicated time periods. ALP activity was measured by an ALP kit, normalized to the cellular protein contents, and presented as mean ±standard deviation (SD) (n = 3). (B, C) Effect of ghrelin on Runx2 expression. The cells were cultured for 48 h with or without 10−6 mol/L ghrelin and presented as mean ±SD (n = 3). The expression of Runx2 was measured by western blotting. (D) Effect of ghrelin on BMP-2 mRNA expression. The cells were cultured with or without 10−6 mol/L ghrelin for the indicated time periods. BMP-2 mRNA expression was determined by real-time quantitative polymerase chain reaction (qPCR). Results are expressed as fold of control. Bars present mean ±SD (n = 3). (E) A representative entire plate view of the Alizarin Red S staining in 24-well plates for control cells and cells treated with ghrelin in 12-day cultures. (F) Effect of ghrelin on calcium deposition. The cells were cultured for 12 days with or without 10−6 mol/L ghrelin and presented as mean ±SD (n = 3). The calcium contents of the cell layers were measured by the atomic absorption spectroscopy method.