Skip to main content
. 2020 May 7;40(7):1680–1694. doi: 10.1161/ATVBAHA.119.313765

Figure 4.

Figure 4.

FOXO1 (forkhead box O1 protein) inhibitor prevents calcification via reducing ALPL promoter activation. A, RT-qPCR analysis of ALPL mRNA isolated from cells in osteogenic conditions receiving 1 µM AS-184856 for 5 d. Results representative of n=5 per group using 3 control and 3 arterial calcification due to deficiency of CD73 (ACDC) patient cell lines. **P=0.0077, ##P=0.0039. Two-way ANOVA with Tukey multiple comparison test was used for statistical analysis. B, TNAP activity staining in cells cultured for 5 d in osteogenic media supplemented daily with the FOXO1 inhibitor AS-1842856 (1 µM) or vehicle control. Results representative of n=5 per group using 4 control and 3 ACDC patient cell lines. Scale bar represents 200 µm. C, Alizarin Red S stain in cells cultured under osteogenic conditions for 21 d and supplemented 1 µM AS-1842856. Results representative of n=6 per group using 4 control and 3 ACDC patient cell lines. ****P>0.0001 by 2-way ANOVA with Tukey multiple comparison test. Scale bar represents 200 µm. D, Luciferase assay of plasmid DNA containing the Gaussia luciferase under control of the human ALPL promoter (hALPL). Cells were transfected with 1 µg of plasmid and cultured for 5 d under osteogenic stimulation. Results representative of n=3 per group using 3 control and 3 ACDC patient cell lines. ***P=0.0011 by 2-way ANOVA with Sidak multiple comparisons test. E, Luciferase assay of hALPL plasmid in cells treated with 100 µmol/L AMP with and without pretreatment of 200 nM rapamycin. Results representative of n=3 using 1 ACDC patient cell line. *P=0.0406. F, Luciferase assay of hALPL and Luciferase assay of hALPL with site-directed mutagenesis (SDM) of the putative FOXO1-binding site. Results representative of n=3 using 1 ACDC patient cell line. ***P=0.0008. Unpaired t test with Welch correction was used for E and F.