Research Paper Volume 13, Issue 8 pp 11610—11628

circACTA2 mediates Ang II-induced VSMC senescence by modulation of the interaction of ILF3 with CDK4 mRNA

ILF3 inhibits VSMC senescence by binding and stabilizing CDK4 mRNA. (A) qRT-PCR detection of CDK4 mRNA expression in VSMCs transfected with ILF3-expressing vector (oeILF3) or shILF3 and their corresponding control. Data represent the means ± SEM of 3 independent experiments. *P B) VSMCs were transfected with shILF3 or oeILF3 and their corresponding control, and then exposed to actinomycin D for 0, 2, 4, and 8 h. CDK4 mRNA level was detected by qRT-PCR. *P C) VSMCs were transfected with oeILF3 or empty vector. ILF3 in the anti-ILF3 immunoprecipitates was measured by Western blotting with anti-ILF3 antibody. (D) GST pull-down of the indicated mRNAs with recombinant GST-ILF3 or GST from the lysates of VSMCs. The cyclin E1, CDK4 and cyclin D1mRNA on the beads was subjected to qRT-PCR detection. (E) The lysates of VSMCs were pulled down with cyclin D1, cyclin E1 or CDK4 3′ UTR probes, and ILF3 in the precipitates was detected by Western blot analysis. (F, H) RIP-PCR detected the CDK4 mRNA and ILF3 interaction in VSMCs treated with or without Ang II (F) or in artery tissues of hypertensive patients (H). *P **P G, I) Probe of CDK4 mRNA was used to detect ILF3 and CDK4 mRNA interaction in VSMCs treated with or without Ang II (G) or in artery tissues of hypertensive patients (I).

Figure 4. ILF3 inhibits VSMC senescence by binding and stabilizing CDK4 mRNA. (A) qRT-PCR detection of CDK4 mRNA expression in VSMCs transfected with ILF3-expressing vector (oeILF3) or shILF3 and their corresponding control. Data represent the means ± SEM of 3 independent experiments. *P < 0.05 vs. their corresponding control. (B) VSMCs were transfected with shILF3 or oeILF3 and their corresponding control, and then exposed to actinomycin D for 0, 2, 4, and 8 h. CDK4 mRNA level was detected by qRT-PCR. *P < 0.05 vs. shCtrl or empty vector. (C) VSMCs were transfected with oeILF3 or empty vector. ILF3 in the anti-ILF3 immunoprecipitates was measured by Western blotting with anti-ILF3 antibody. (D) GST pull-down of the indicated mRNAs with recombinant GST-ILF3 or GST from the lysates of VSMCs. The cyclin E1, CDK4 and cyclin D1mRNA on the beads was subjected to qRT-PCR detection. (E) The lysates of VSMCs were pulled down with cyclin D1, cyclin E1 or CDK4 3′ UTR probes, and ILF3 in the precipitates was detected by Western blot analysis. (F, H) RIP-PCR detected the CDK4 mRNA and ILF3 interaction in VSMCs treated with or without Ang II (F) or in artery tissues of hypertensive patients (H). *P < 0.05, **P < 0.01 vs. their corresponding control. (G, I) Probe of CDK4 mRNA was used to detect ILF3 and CDK4 mRNA interaction in VSMCs treated with or without Ang II (G) or in artery tissues of hypertensive patients (I).