Research Paper Volume 13, Issue 10 pp 13859—13875

Figure 3. LCN2 is essential for the inhibition of VSMCs proliferation by CMKLR1 depletion. (A) IHC analysis of Lipocalin-2 (LCN2) expression in aorta tissues of mice fed with normal and HFD. Scale bar = 100 μm. Red arrows indicate plaques. (B) Chemerin expression score analysis data presented as the means ± SDs. **, p< 0.01. (C) Western blots showing LCN2, MMP-9, and β-catenin expression in cells treated with chemerin-9 (48 h) for different durations. (D) Western blot showing LCN2, MMP-9, and β-catenin expression, with or without CMKLR1 knockdown. (E) mRNA expression of LCN2, with or without CMKLR1 depletion, as determined by quantitative real-time PCR. (F) Western blots showing CMKLR1 and LCN2 expression following the application of plasmids encoding CMKLR1 to overexpress CMKLR1 expression in VSMCs with or without CMKLR1 depletion. (G) Western blots showing CMKLR1, LCN2, PCNA and MMP-9 expression following the application of plasmids encoding LCN2 to overexpress LCN2 expression in VSMCs with or without CMKLR1 depletion. (H) Analysis of the percentage EdU-incorporated cells. ****, p< 0.0001; ns, no significant difference. (I) Cell migration as determined by wound healing assay. Percentage of cell migration data presented as means ± SDs. ****, p< 0.0001; **, p< 0.01; *, p< 0.05. (J) Western blots showing CMKLR1, LCN2, p-AKT, AKT, p-ERK 1/2, ERK 1/2, p-p38, p38, p-PKC and β-catenin expression following the application of plasmids encoding LCN2 to overexpress LCN2 expression in VSMCs with or without CMKLR1 depletion. (K) Western blots showing CMKLR1, LCN2, p-p38, p38 and β-catenin expression following the transfection of small interfering RNA (siRNA) targeting LCN2 (siLCN2) into VSMCs with or without CMKLR1 overexpression.