Skip to main content
. 2021 Jul 15;131(14):e146985. doi: 10.1172/JCI146985

Figure 3. ALKBH1-demethylated DNA 6mA modification is reduced in murine calcified arteries.

Figure 3

(A) Leukocyte DNA 6mA level in mice fed with adenine diet (CKD, n = 13) or normal chow diet (control, n = 12) for 8 weeks. Leukocytes were isolated from peripheral blood. (B and C) Mice leukocyte DNA 6mA level in different subgroups defined by the percentage of calcification lesion size in aortic smooth muscle layer (B, n = 12 for non-VC; n = 6 for mild; n = 7 for severe). Scatter dot plot of correlation between mice leukocyte DNA 6 mA level and percentage of calcification lesion size in aortic smooth muscle layer from mice fed with adenine diet for 8 weeks (C, n = 13). (D) The mRNA expression levels of Alkbh1 and N6amt1 in leukocytes from mice with different diets (n = 12 per group). (E) Representative immunohistochemistry pictures and quantification of ALKBH1, N6AMT1, and 6mA in mice aortic smooth muscle layer (n = 10 for control; n = 12 for CKD). Scale bars: 50 μm. (F) Western blot analysis of ALKBH1 and N6AMT1 expression in mice aortic arch (n = 4 for control; n = 5 for CKD). (G–I) Calcium content (G), Western blot analysis of ALKBH1 and N6AMT1 (H), and DNA 6 mA level (I) in mice aortic rings incubated with osteogenic medium for the indicated time (0, 3, 5, 7, 10, and 14 days) (n = 4–6 per group). Statistical significance was assessed using 2-tailed t tests (A and D–F), 1-way ANOVA followed by Bonferroni’s test (B) or Dunnett’s test (G–I), and Pearson’s correlation coefficient analysis (C). All values are presented as mean ± SD. *P < 0.05 vs. Pi (0 day) in (G–I).