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. 2021 Apr 15;9(4):427. doi: 10.3390/biomedicines9040427

Figure 5.

Figure 5

The effect of bilirubin, iron, and CO on the calcification of VICs. (a) Schematic of HO-1-catalyzed heme degradation. (b–g) VICs (passage number 5–8) were maintained in GM, OM, or OM supplemented with (b,e) bilirubin (BR; 5-50 µmol/L), (c,f) iron (Fe; 5-50 µmol/L), or (d,g) CO donor (CORM; 5-50 µmol/L) for 5 days. (b–d) Ca deposition in the ECM was evaluated after 5 days by AR staining. Representative images of stained plates from three independent experiments and quantification are shown. (e–g) Ca content of HCl-solubilized ECM is shown. (h–j) VICs were maintained in GM, OM, or OM supplemented with heme (H, 25 µmol/L), BR (50 µmol/L), Fe (50 µmol/L), or CORM (50 µmol/L). (h) OCN levels were determined in EDTA-solubilized ECM samples by ELISA (day 5). (i,j) SOX9 and OPN mRNA levels were measured by real time RT-PCR (72 h). Data are expressed as mean ± SD of three independent experiments performed in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.