Fig. 4

IP-10/IL-17 co-treatment-initiated HCASMC calcification is mediated by the BMP6 autocrine effect. a The HCASMCs were either maintained as the control or were co-treated with IP-10 and IL-17 for 3 days, after which we examined the BMP2/4/6 mRNA expression in the HCASMCs; b–d the HCASMCs were either maintained as the control or were pre-treated with IgG, BMP2-, BMP4-, or BMP6-blocking antibodies and then co-treated with IP-10 and IL-17; b HCASMC calcification was analyzed using ARS stain; c we examined the mRNA expressions OPN, OCN, and ALP using real-time PCR; d the protein expressions of OPN, OCN, and ALP were examined using the western blot test; e the HCASMCs were either maintained as the control or were treated with IP-10, IL-17, or both, for 48, 72, or 96 h, after which we examined the smad1/5 phosphorylation using the western blot test; f the HCASMCs were pretreated with control-, smad1- or smad5-specific siRNA and then either maintained as the control or co-treated with IP-10 and IL-17 for 14 days. The calcification of the HCASMCs was analyzed using ARS stain. The data in (a–f) are mean ± SEM from three independent experiments. * P < 0.05 vs. control cells; # P < 0.05 vs. IgG/or si-CL/IP-10 and IL-17-co-treated cells. The results in (d–e) are representative of three independent experiments with similar results