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

circACTA2 interacts with ILF3 in VSMCs. (A) Schematic of the experimental design based on RNA antisense purification (RAP) or U-biotin-labeled RNA pull-down followed by LC-MS/MS analysis. (B) Western blot detection of FN1, VIM and ILF3 in the circACTA2 probe-pulled down precipitates (circACTA2-overexpressed group vs. control group). (C) qRT-PCR detection of circACTA2 level in the RNA-protein immunoprecipitates pulled down by anti-FN1, anti-VIM and anti-ILF3 antibodies. *P **P D) Western blot detection of ILF3 and VIM expression in VSMCs transfected with empty vector or circACTA2. (E) VSMCs were transfected with circACTA2 and treated with or without Ang II, and then cell lysates were pulled down with circACTA2 probe and detected by western blotting with anti-ILF3 and anti-VIM antibodies. (F, H) RIP-PCR detected the circACTA2 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 circACTA2 was used to detect ILF3 and circACTA2 interaction in VSMCs treated with or without Ang II (G) or in artery tissues of hypertensive patients (I).

Figure 2. circACTA2 interacts with ILF3 in VSMCs. (A) Schematic of the experimental design based on RNA antisense purification (RAP) or U-biotin-labeled RNA pull-down followed by LC-MS/MS analysis. (B) Western blot detection of FN1, VIM and ILF3 in the circACTA2 probe-pulled down precipitates (circACTA2-overexpressed group vs. control group). (C) qRT-PCR detection of circACTA2 level in the RNA-protein immunoprecipitates pulled down by anti-FN1, anti-VIM and anti-ILF3 antibodies. *P < 0.05, **P < 0.01 vs. IgG. (D) Western blot detection of ILF3 and VIM expression in VSMCs transfected with empty vector or circACTA2. (E) VSMCs were transfected with circACTA2 and treated with or without Ang II, and then cell lysates were pulled down with circACTA2 probe and detected by western blotting with anti-ILF3 and anti-VIM antibodies. (F, H) RIP-PCR detected the circACTA2 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 circACTA2 was used to detect ILF3 and circACTA2 interaction in VSMCs treated with or without Ang II (G) or in artery tissues of hypertensive patients (I).