AVS-patient HAVIC calcification induced by MK-4 in high-Pi medium. HAVICs from patients with AVS were cultured in α-MEM containing 10% FBS, and at 80%–90% confluency (day 0), the cells were further cultured for 7 days. (A) The quantification on day 7 of Alizarin Red S staining of AVS-patient HAVICs after cetyl-pyridinium chloride extraction. The amount of released dye was quantified by measuring the 550-nm absorbance. All ratios were normalized to untreated control values on day 7. White bar: untreated cells; black bar: cells treated with high Pi (3.2 mM); light-blue bar: cells treated with PFA (0.1 mM) in normal Pi (1.0 mM); light-red bar: cells treated with MK-4 (10 nM) in normal Pi; red bar: cells treated with MK-4 in high Pi; blue bar: cells treated with MK-4 and PFA in high Pi. Bars represent means ± S.E.M. (n = 5). One-way ANOVA with Tukey's multiple comparisons test. (B) BMP2 gene expression and (C) ALP activity in HAVICs from patients with AVS were measured on day 7. All ratios were calculated versus the control group on day 0. Relative gene-expression levels were determined by normalizing measured values to those obtained for the housekeeping gene G3PDH. White bar: untreated cells; black bar: cells treated with high Pi (3.2 mM); light-red bar: cells treated with MK-4 (10 nM) in normal Pi (1.0 mM); red bars: cells treated with MK-4 and high Pi. Bars represent means ± S.E.M. (n = 5 (B), n = 4 (C)). One-way ANOVA with Tukey's multiple comparisons test. (D) HAVIC calcification induced by MK-4 in the presence or absence of Smad1/5/8 phosphorylation inhibitor dorsomorphin (Dorso) or NF-κB inhibitor SN-50 in high-Pi medium. Quantification on day 7 of Alizarin Red S staining after cetyl-pyridinium chloride extraction. The amount of released dye was quantified by measuring the 550-nm absorbance. All ratios were normalized to the control value on day 7. White bar: untreated cells; red bar: cells treated with 10 nM MK-4 and high Pi (3.2 mM); light-cyan blue bar: cells treated with high Pi and 3 μM Dorso, an inhibitor of Smad1/5/8 phosphorylation; cyan blue bar: cells treated with high Pi, MK-4, and 3 μM Dorso; light-green bar: cells treated with high Pi and 10 μM SN-50, an NF-κB inhibitor; green bar: cells treated with high Pi, MK-4, and SN-50. Bars represent means ± S.E.M. (n = 5). One-way ANOVA with Tukey's multiple comparisons test.