Porcine aortic VICs were cultured in Osteogenic Media (OM) or Control Media (CM) for 9 days, ± BMP inhibitor LDN-193189. OM-induced calcific nodules can be observed by Alizarin Red (red, C) and von Kossa (brown, G) staining, and formation of these nodules is inhibited by LDN-193189 (D,H). The total number of calcific nodules for each treatment group, as detected by Alizarin Red (I) and von Kossa (J) staining, was quantified. Each dot is representative of the total number of calcific nodules quantified per well for each treatment group. Error bars are shown as interquartile means and scatter plot. Statistical significance was determined by Mann-Whitney U-test (p< 0.05) for comparisons indicated by lines.