Figure 6. Kynurenine promotes ubiquitin-mediated proteasomal degradation of RUNX2 in VSMCs.

A, Quantitative RT-PCR analysis of RUNX2 expression in human coronary VSMCs transfected with scramble (Scr) siRNA or IDO1 siRNA (left panel), and human coronary VSMCs treated with DMSO or kynurenine (Kyn, 50 μmol/L) for 48 hours (right panel) (n = 6).

B, Quantitative RT-PCR analysis of RUNX2 expression in human coronary VSMCs transfected with scramble (Scr) siRNA or IDO1 siRNA (left panel), and human coronary VSMCs treated with DMSO or Kyn (50 μmol/L) for 48 hours, followed by treatment with actinomycin D (Act D, 1 μmol/L) for different periods (right panel) (n = 4–6).

C-E, Western blot analysis of RUNX2 expression in human coronary VSMCs transfected with Scr siRNA or IDO1 siRNA, and then treated with chloroquine (CQ, 20 μmol/L) (C), MG132 (1 μmol/L) (D), or cycloheximide (CHX, 50 μg/mL) (E) for the indicated periods (n = 4).

F, Western blot analysis of RUNX2 expression in RUNX2-overexpressed human coronary VSMCs treated with DMSO or Kyn (50 μmol/L), followed by treatment with CHX (50 μg/mL) for different periods (n = 4).

G and H, Human coronary VSMCs with Scr siRNA or IDO1 siRNA transfection (G), DMSO or Kyn (50 μmol/L) supplementation (H) were pretreated with MG132 (1 μmol/L). The expression of RUNX2, ubiquitin (Ub), and GAPDH was determined by Western blot analysis (Input). Immunoprecipitation was performed with RUNX2 or IgG antibody, RUNX2 and RUNX2-bound Ub was determined by Western blot analysis (IP; n = 3).

Results are presented as mean ± SEM. P values are assessed using two-tailed unpaired Student’s t-test used for A; Two-way ANOVA with Tukey’s post hoc test is used for B-F. ***P < 0.001, ns indicates P > 0.05.