Figure 5. circACTA2 promotes VSMC senescence by competing with CDK4 mRNA to bind to ILF3. (A) circACTA2, cyclin D1 and CDK4 mRNAs were pulled down with recombinant GST-ILF3 from the lysates of VSMCs treated with or without Ang II for 3 days. The cyclin D1 and CDK4 mRNA as well as circACTA2 on the beads were subjected to qRT-PCR detection. **P < 0.01, ***P < 0.001 vs. vehicle control. (B) The lysates of VSMCs treated with or without Ang II were pulled down with CDK4 3′ UTR or circACTA2 probe, and ILF3 in the precipitates was detected by Western blot analysis. (C) VSMCs were transfected with circACTA2 and ILF3-expressing vector (oeILF3) either alone or together. Then cells were exposed to actinomycin D for 0, 2, 4, and 8 h. CDK4 mRNA level was detected by qRT-PCR. *P < 0.05 vs. their corresponding control. (D) SA-β-gal activity in VSMCs transfected as in (C). The percentage of SA-β-gal positive cells (bottom) and representative pictures (top) are shown. Magnification × 400. *P < 0.05, **P < 0.01 vs. their corresponding control. (E) VSMCs were transfected as in (C), and the expression of CDK4 and ILF3 was examined by immunofluorescence staining. Green, red, and blue staining indicates CDK4, ILF3, and the nuclei, respectively. Scale bar = 100 μm.