图1 人冠状动脉实验

Fig.1 Examinations of human coronary arteries. A: Human coronary artery tissue specimens. B: Microscopic observation of coronary atherosclerosis (AS; HE staining, scale bar=1 mm). C: Partial co-localization of ATF3 (red) and CD68 (green) in the plaques (arrow,scale bar=50 μm). D: Western blotting for detecting ATF3 and CD68 expressions in the plaques. All experiments were repeated at least 3 times, and data are presented as Mean±SD (n=20). *P<0.05 vs Con group.

图2 ApoE-/-小鼠ATF3基因敲除模型鉴定

Fig.2 Verification of ATF3 gene knockout in ApoE-/- mice. A: Schematic diagram of mouse grouping and treatment. B: Observation of a mouse aorta. The yellow lines indicate the sampling site, and the red box indicates the tissues used for preparing frozen sections or paraffin sections. C: Expression of eGFP in the aortic vessels. After masking autofluorescence in the mouse aortas with pontamine sky blue, no fluorescence signals were detected in the vessels in the control group, but eGFP expression (arrows) was observed in the aortic plaques in AAV9-eGFP group. D: Immunohistochemical staining for detecting distribution of ATF3 expression in the aorta (a1-d1 are enlarged images of the boxed areas in a-d. Scale bar=50 μm, n=6). E: Western blotting for detecting aortic ATF3 protein content (n=6). Data are presented as Mean±SD. *P<0.05 vs Con group; #P<0.05 vs AS or AAV9-eGFP group.

图3 ATF3基因敲除后ApoE-/-小鼠主动脉斑块改变

Fig.3 Changes in mouse aortic plaques after ATF3 gene knockout. A: Gross oil red O staining of the aorta showing lipid deposition in the total vascular area (n=6). B: Oil red O staining of cross-sections within the plaque area showing lipid deposition in the aortic lumen (n=3). C: HE staining of the aortic plaque area (n=6). Scale bar=50 μm. Data are presented as Mean±SD. *P<0.05 vs Con group, #P<0.05 vs AS or AAV9-eGFP group.

图4 ATF3基因敲除后斑块内炎症因子表达增强

Fig.4 ATF3 gene knockout increases expressions of inflammatory factors in aortic plaques of the mice. A: Immunohistochemical staining for detecting aortic CD45, CD68, IL-1β and TNF-α protein expressions and MOD value statistics (scale bar=50 μm, n=6). B: Western blotting of aortic CD45, CD68, IL-1β and TNF-α protein expressions (n=6). Data are presented as Mean±SD. *P<0.05 vs Con group, #P<0.05 vs Con, AS or AAV9-eGFP group.

图5 ATF3基因敲除后斑块内VCAM1、MMP-2和MMP-9蛋白增强

Fig. 5 ATF3 knockout increases expressions of VCAM1, MMP-2 and MMP-9 in aortic plaques of the mice. A: Immunohistochemical staining for detecting aortic VCAM1, MMP-2 and MMP-9 protein expressions and MOD value statistics (scale bar=50 μm, n=6). B: Western blotting for detecting aortic VCAM1, MMP-2 and MMP-9 protein expressions (n=6). Data are presented as Mean±SD. *P<0.05 vs Con group, #P<0.05 vs AS or AAV9-eGFP group.

图6 ATF3基因敲除后NF-κB信号通路激活

Fig.6 ATF3 knockout activates the NF-κB signaling pathway in aortic plaques of the mice. A: Immunohistochemical staining for detecting aortic p-IKKα/β and p-NF-κB P65 protein expressions and MOD value statistics (scale bar=50 μm, n=6). B: Western blotting for detecting aortic IKKα/β, p-IKKα/β, NF-κB P65 and p-NF-κB P65 expressions (n=6). Data are presented as Mean±SD. *P<0.05 vs Con group, #P<0.05 vs AS or AAV9-eGFP group.

图7 细胞实验结果

Fig.7 Cell experiment results. A: THP-1 cells, macrophages and foam cells stained with oil red O to identify lipid deposition (scale bar=50 μm). B: Western blotting for detecting ATF3 silencing efficiency and IKKα/β, p-IKKα/β, NF-κB P65, and p-NF-κB P65 protein expressions. C: Western blotting for detecting ATF3 overexpression efficiency and IKKα/β, p-IKKα/β, NF-κB P65, and p-NF-κB P65 protein expressions. Data are presented as Mean±SD. *P<0.05 vs PMA group, #P<0.05 vs PMA+Ox-LDL, PMA+Ox-LDL+Lip3000 or PMA+Ox-LDL+OE-vector group.