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 overexpression suppresses Ang II-induced VSMC senescence. (A) Western blot detection of ILF3, p21, p16 and CDK4 expression in VSMCs transfected with shCtrl or shILF3. (B) SA-β-gal activity in VSMCs transfected with shCtrl or shILF3. The percentage of SA-β-gal positive cells (above) and representative pictures (below) are shown. Magnification × 400. *P C) Western blot detection of ILF3, p21, p16 and CDK4 expression in VSMCs transfected with empty vector or ILF3-expressing vector (oeILF3). (D) SA-β-gal activity in VSMCs transfected with empty vector or oeILF3. The percentage of SA-β-gal positive cells (above) and representative pictures (below) are shown. Magnification ×400. *P E) and (F) VSMCs were transfected with shILF3 or oeILF3 and their corresponding controls, and the expression of ILF3, p16 and p21 was examined by immunofluorescence staining. Green, red, and blue staining indicates ILF3, p16 (E), p21 (F) and the nuclei, respectively. Scale bar = 100 μm. (G) VSMCs were transfected as in (E), and cell proliferation was estimated by BrdU incorporation test. Graph presents means ± SD from at least three independent experiments. *P H) Western blot detection of p21 and CDK4 expression in VSMCs transfected with empty vector or oeILF3 followed by treatment with or without Ang II. (I) SA-β-gal activity in VSMCs transfected with empty vector or oeILF3 followed by treatment with or without Ang II. The percentage of SA-β-gal positive cells (bottom) and representative pictures (top) are shown. Magnification × 400. *P **P

Figure 3. ILF3 overexpression suppresses Ang II-induced VSMC senescence. (A) Western blot detection of ILF3, p21, p16 and CDK4 expression in VSMCs transfected with shCtrl or shILF3. (B) SA-β-gal activity in VSMCs transfected with shCtrl or shILF3. The percentage of SA-β-gal positive cells (above) and representative pictures (below) are shown. Magnification × 400. *P < 0.05 vs. shCtrl. (C) Western blot detection of ILF3, p21, p16 and CDK4 expression in VSMCs transfected with empty vector or ILF3-expressing vector (oeILF3). (D) SA-β-gal activity in VSMCs transfected with empty vector or oeILF3. The percentage of SA-β-gal positive cells (above) and representative pictures (below) are shown. Magnification ×400. *P < 0.05 vs. empty vector. (E) and (F) VSMCs were transfected with shILF3 or oeILF3 and their corresponding controls, and the expression of ILF3, p16 and p21 was examined by immunofluorescence staining. Green, red, and blue staining indicates ILF3, p16 (E), p21 (F) and the nuclei, respectively. Scale bar = 100 μm. (G) VSMCs were transfected as in (E), and cell proliferation was estimated by BrdU incorporation test. Graph presents means ± SD from at least three independent experiments. *P < 0.05 vs. their corresponding control. (H) Western blot detection of p21 and CDK4 expression in VSMCs transfected with empty vector or oeILF3 followed by treatment with or without Ang II. (I) SA-β-gal activity in VSMCs transfected with empty vector or oeILF3 followed by treatment with or without Ang II. 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.