图1 ACADM 在乳腺癌中的表达水平及其对 ER 阳性乳腺癌细胞恶性生物学行为的影响

Fig.1 Expression level of ACADM in breast cancer tissues and its effect on malignant behaviors of estrogen receptor (ER)-positive breast cancer cells. A: Expression levels of ACADM detected by Western blotting in normal breast epithelial cells and ER-positive breast cancer cell lines (**P<0.01, ##P<0.01 vs MCF-10A cells). B: Prognosis of breast cancer patients with low and high ACADM expression obtained from the Kaplan-Meier Plotter bioinformatics website. C: Expression of ACADM protein in MCF-7 and T47D cells were detected by Western blotting after down-regulation of ACADM (*P<0.05, **P<0.01 vs NC). D: CCK8 assay for assessing proliferation of MCF-7 cells after down-regulation of ACADM. E: Changes of tumor size of in situ MCF-7 cell xenograft with ACADM knockdown in nude mice. F: HE staining of hepatic metastases in nude mice with tail vein injection MCF-7 cells with or without ACADM knockdown (Original magnification:×200). G: Western blotting for detecting protein expressions in the EMT signaling pathway in MCF-7 cells with ACADM knockdown. *P<0.05, **P<0.01, ***P<0.001 vs NC.

图2 下调 ACADM 表达后 MCF-7 细胞和 T47D 细胞的脂肪毒性影响细胞的侵袭能力

Fig.2 Down-regulation of ACADM-mediated lipotoxicity attenuates invasiveness of MCF-7 and T47D cells. A: Lipid droplets in shACADM MCF7 and T47D cells detected by Oil red O staining (×100). B: Free fatty acid content in shACADM MCF7 and T47D cells detected using a microplate reader (**P<0.01, ***P<0.001 vs NC). C: Ester CoA contents are increased in shACADM MCF-7 and T47D cells (*P<0.05 vs NC). D:Transwell assay for assessing invasiveness of MCF-7 and T47D cells with ACADM knockdown cultured in 4% serum (**P<0.01 vs shACADM). E: Boyden chamber assay for assessing migration ability of MCF-7 and T47D cells with ACADM knockdown cultured in 4% serum (*P<0.05, **P<0.01 vs shACADM).

图3 下调 ACADM 导致 ROS 增加影响细胞侵袭能力

Fig.3 ACADM knockdown results in increased levels of ROS in breast cancer cells. A: ROS levels in MCF-7 and T47D cells with ACADM knockdown detected by fluorescence microscopy (×100). B: ROS levels in MCF-7 and T47D cells with ACADM knockdown detected by flow cytometry (***P<0.001, ****P<0.0001 vs NC). C: Lipid oxidation (MDA) in MCF-7 and T47D cells with ACADM knockdown detected by colorimetric method (**P<0.01 vs NC). D: ROS content in MCF-7 and T47D cells with ACADM knockdown detected by flow cytometry after treatment with ROS scavenger (****P<0.0001 vs NC; ##P<0.01, ####P<0.0001 vs shACADM). E: Invasion and migration of MCF-7 and T47D cells with ACADM knockdown detected by Transwell assay after treatment with ROS scavengers (**P<0.01, ***P<0.001 vs NC; #P<0.05, ##P<0.01 vs shACADM). F: Invasion and migration of MCF-7 and T47D cells with ACADM knockdown detected by Boyden chamber assay after treatment with ROS scavengers (**P<0.01, ***P<0.001 vs NC; #P<0.05, ###P<0.001 vs shACADM).

图4 下调ACADM通过损害线粒体功能影响细胞侵袭转移能力

Fig.4 Downregulation of ACADM impairs mitochondrial function in breast cancer cells. A: Seahorse technique for detecting oxygen consumption in MCF-7 cells with ACADM knockdown. B: Glycolysis in MCF-7 cells with ACADM knockdown detected using Seahorse technique. C: ATP content in MCF-7 and T47D cells with ACADM knockdown (*P<0.05 vs NC). D: Mitochondrial ROS in MCF-7 and T47D cells with ACADM knockdown detected by confocal fluorescence microscopy (×100). E: Content of mitochondrial ROS in MCF-7 and T47D with ACADM knockdown detected by flow cytometry (***P<0.001, ****P<0.0001 vs NC). F: Activity of mitochondrial respiratory chain complex III in shACADM MCF-7 and T47D cells (*P<0.05 vs NC). G: Activity of mitochondrial respiratory chain complex V in shACADM MCF-7 and T47D cells (*P<0.05, **P<0.01 vs NC).

图5 ACADM通过线粒体心磷脂影响细胞侵袭能力

Fig.5 ACADM knockdown reduces mitochondrial cardiolipin to attenuate invasion and migration of breast cancer cells. A: Hierarchical clustering of lipid species in shACADM MCF-7 and T47D cells. B: Contents of mitochondrial ROS detected by flow cytometry in shACADM MCF-7 and T47D cells treated with Elamipretide (****P<0.0001 vs NC; ###P<0.001, ####P<0.0001 vs shACADM). C: Invasion and migration ability of Elamipretide-treated shACADM MCF-7 and T47D cells detected by Transwell assay (**P<0.01 vs NC; #P<0.05, ##P<0.01 vs shACADM). D: Invasion and migration ability of Elamipretide-treated shACADM MCF-7 and T47D cells detected by Boyden chamber assay (**P<0.01 vs NC; #P<0.05, ##P<0.01 vs shACADM).

图6 ACADM通过调控PI3K/AKT通路影响细胞侵袭能力

Fig.6 ACADM knockdown affects cell invasion capacity by regulating the PI3K/AKT pathway. A: Expressions of PI3K-AKT pathway proteins in shACADM MCF-7 cells and T47D cells detected by Western blotting (*P<0.05, **P<0.01 vs NC). B: Expression of p-AKT protein in shACADM MCF-7 cells and T47D cells with SC79 treatment detected by Western blotting (*P<0.05, **P<0.01 vs NC; #P<0.05, ###P<0.001 vs shACADM). C: Invasion and metastasis ability of shACADM MCF-7 and T47D cells detected by Transwell assay after SC79 treatment (*P<0.05, **P<0.01vs NC; #P<0.05, ##P<0.01 vs shACADM). D: Invasion and migration ability of shACADM MCF-7 and T47D cells detected by Boyden chamber assay after SC79 treatment (*P<0.05 vs NC; #P<0.05, ##P<0.01 vs shACADM). E: ROS levels in shACADM MCF-7 and T47D cells detected by fluorescence microscope after ROS inhibitor treatment (×100). F: Expressions of PI3K-AKT pathway proteins in shACADM MCF-7 cells and T47D cells treated with ROS inhibitor detected by Western blotting (*P<0.05 vs NC; #P<0.05, ##P<0.01 vs shACADM).