Figure 1. SMC-Klf4 KO resulted in a marked reduction in overall lesion senescence, including reduced expression of the prosenescence markers p21⁺ and SAβG⁺ cells.

(A) Experimental design, SMC (Myh11-CreER^T2-eYFP) lineage tracing Klf4 WT versus KO Apoe^–/– mice were injected with tamoxifen at 6–8 weeks of age and subsequently placed on western diet (WD) for 26 weeks to induce advanced atherosclerosis. (B) SAβG staining was performed as described in Methods on freshly isolated aortas. Thoracic and abdominal aortic sections from SAβG-stained aortas from the left images and counter stained with nuclear fast red and yellow arrows indicate the SAβG⁺ cells. Original magnification ×10. (C) Quantification of SAβG⁺ area of the aorta. (D) Representative images of costaining for eYFP (for detecting SMC), α-SMA, p21 (a marker of senescence), and DAPI (nucleus) in advanced BCA lesions from SMC Klf4 WT and KO animals fed a WD for 26 weeks. The confocal images show a maximum intensity projection ×20 zoom. Scale bar: 100 μm. (E and F) Quantification of the frequency of p21⁺ (p21⁺/DAPI) senescent cells in the lesion (E) and fibrous cap (F) as a percent of total cells in the lesions. (G and H) Quantification of SMC derived p21⁺ (eYFP⁺ p21⁺/eYFP) senescent cells in the lesion (G) and the fibrous cap (H) as a percent of total SMC. (I) Quantification of α-SMA⁺ senescent (α-SMA⁺ p21⁺/α-SMA) cells in the lesion as a percentage of total α-SMA⁺ cells. Mann-Whitney U tests were used for statistical analysis in C and E–I. Data are shown as mean ± SEM. Independent animals are indicated as individual dots (WT, n = 7, and KO, n = 9). The P values are indicated on the respective graphs.