Figure 6. Pyrophosphate generation and ENPP expression are enhanced in VIC by iron, apo-ferritin and D3T.

A) VIC was cultured in growth or calcification medium supplemented with 50 μmol/L iron (Ammonium iron (III) citrate), 2 mg/mL apo-ferritin or 75 μmol/L D3T for five days and pyrophosphate level was measured. B) VIC was transfected with siRNA specific to H-ferritin or negative control siRNA prior to iron exposure and pyrophosphate level was measured. C) ENPP2 expression in VIC was assessed by Western blot analysis. D) ENPP2 Western blot were shown. AS tissues derived VIC cells were cultured in calcific condition alone or treated with H-ferritin. Samples were normalized to the protein content of the cells. E) PPi level changes after H-Ferritin silencing were shown. VIC cells were transfected with siRNA against H-Ferritin. Samples were treated in the next day with 1 mg/mL of H-Ferritin and 1 mg/mL H-Ferritin 222 (mutant ferritin without ferroxidase activity) for 3 days. Data were analyzed by One Way ANOVA, Bonferroni’s Multiple Comparison Test and are shown as mean values ± SEM of three separate experiments performed in duplicate. Not significant (ns), *P < 0.05; ***P < 0.0001.