Fig. 1: MLN4924 inhibits Pi-induced vascular calcification.

a Treatment with MLN4924 (0.001 µM, 0.01 µM, 0.1 µM, or 1 µM) inhibited Pi (4 mM)-induced calcium deposition in A10 cells. n = 4–12 per group, independent experiments. b A10 cells were treated with Pi and MLN4924 under the aforementioned conditions. Representative alizarin red S-stained VSMCs showing that mineralization was blocked by MLN4924 in Pi-treated A10 cells. Scale bar, 10 mm. c Quantification of alizarin red S staining. n = 6 per group, independent experiments. d mRNA levels of smooth muscle marker genes (SM22α and SMA) and osteogenic-related marker genes (RUNX2 and ALP) were measured in A10 cells treated with Pi and MLN4924. n = 6 per group, independent experiments. e NEDD8 immunoblotting was performed after A10 cells were treated with Pi and MLN4924. The red arrow indicates free NEDD8. f, g The amount of accumulated calcium was measured. Overexpression of NEDD8 potentiated Pi-induced calcium deposition in A10 cells. n = 8 per group, independent experiments (f). Inhibition of NEDD8 conjugation with NEDD8-ΔGG blunted Pi-induced calcium deposition. n = 8 per group, independent experiments (g). The data are shown as the means ± SEMs. Statistical significance was tested via ANOVA with Tukey’s HSD test and Dunnett’s T3 test.