Figure 2. SMC-Klf4 KO resulted in a marked reduction of SMC-derived and non–SMC-derived LGALS3⁺ cells also positive for the senescence marker γ-H2AX.

(A) Representative images of costaining for eYFP (for detecting SMC), γ-H2AX (a marker of senescence), LGALS3, and DAPI (nucleus) in advanced BCA lesions from SMC Klf4 WT and KO animals fed WD for 26 weeks as shown in Figure 1A. The confocal images show a maximum intensity projection ×20 zoom. Scale bar: 100 μm and 20 μm (zoomed-in images region of interest). (B) Quantification of the frequency of H2AX⁺ (H2AX⁺/DAPI) senescent cells in the fibrous cap. (C) Myh11-eYFP⁺/DAPI in the fibrous cap. (D) SMC-derived γ-H2AX⁺ cells of all SMC (Myh11-eYFP⁺ γ-H2AX⁺/eYFP) in the fibrous cap. (E) Quantification of SMC-derived γ-H2AX⁺ LGALS3⁺ (Myh11-eYFP⁺ γ-H2AX⁺ LGALS3⁺/eYFP) of all SMC in the fibrous cap. (F) Quantification of non–SMC-derived γ-H2AX⁺ LGALS3⁺ (Myh11-Eyfp^– γ-H2AX⁺ LGALS3⁺/eYFP^–) cells of all non-SMC cells in the fibrous cap. Mann-Whitney U tests were used for statistical analysis in B–F. 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.