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. Author manuscript; available in PMC: 2021 Aug 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2020 Jun 25;40(8):1854–1869. doi: 10.1161/ATVBAHA.120.314458

Figure 3. Pro-differentiation effects of 5-azacytidine in vitro are mediated through PTEN induction.

Figure 3.

SMCs stably expressing control non-targeting shRNA or PTEN targeting shRNA were cultured in 0.1% CS EMEM. (A) SMCs were treated with 5-azacytadine for 24 hrs and RNA was harvested for RT-qPCR analysis of PTEN mRNA. 10 μM 5-azacytidine induced PTEN upregulation in control shRNA SMCs, but induction is blocked in SMCs stably expressing PTEN shRNA. ANOVA with Bonferroni post hoc; **p<0.01; ****p<0.0001. (B) SMCs were treated as in (A) and RT-qPCR used to analyze the indicated genes. Compared to controls, 5-azacytidine was unable to upregulate the differentiation-associated genes, Acta2 and Tet2, in PTEN depleted SMCs. In contrast, while 5-azacytidine blocked basal expression of the inflammatory genes Ccl2 and Ccl7 in control shRNA SMCs, the increased basal expression in PTEN depleted SMCs was not reversed. (C) SMCs were treated with 5-azacytidine and PDGF-BB for 24 hrs and protein was harvested for Western analysis of PTEN. Western blots (left) showing PTEN protein expression in control shRNA SMCs or PTEN KD SMCs following treatment with 5-azacytidine and PDGF-BB for 24 hrs. β-actin is shown as a loading control. (Right) Quantification of PTEN band intensity, normalized to β-actin expression. Fold change is expressed relative to control shRNA SMCs stimulated with vehicle.