Fig 2: LASP1 is associated with sphingomyelin metabolism and regulates ECHS1 in CRC cells.A GSEA of the top 100 upregulated LASP1-related pathways, including biocarta ceramide, PID_S1P_S1P2 and PID_S1P_META. B Heatmap of the LC–MS assay for RKO-LASP1 and control cells. C Heat map depicting the differentially expressed lipomics for RKO-LASP1 and control cells. D Histogram of c. The asterisk (*) indicates P < 0.05. The double asterisk (**) indicates P < 0.01. The quadra asterisk (****) indicates P < 0.0001. E Fluorescence images show the two differentially expressed protein spots in DIGE analysis. The interacting protein spots are indicated (white arrows). F MS of in-gel trypsin digests of the protein and analysis of the depicted peptide spectrum resulted in the identification of ECHS1. G, H RT-PCR (G) and Western blotting (H) were performed to detect the expression of ECHS1 and LASP1 in LASP1-overexpressing and LASP1-knockdown cells. I Representative figures and data of the Transwell assay for LV-control-, LV-LASP1-, and LV-LASP1 plus siRNA-ECHS1-transfected RKO cells. Bars in the right panel represent the number of migrated and invaded cells.

Fig 3: ECHS1 resists autophagy and apoptosis of CRC cells through the PI3K-Akt-mTOR pathway mediated by ceramide.A Western blot analysis of the expression of PI3K/Akt/mTOR pathway members (PI3K, Akt, phosphorylated AKT at Ser473, and phosphorylated mTOR) in ECHS1-overexpressing or ECHS1-knockout CRC cells. B Western blot analysis of the expression of PI3K/Akt/mTOR pathway members in ECHS1-overexpressing cells with the application of the inhibitor of UGCG eliglustat (left panel) or in ECHS1-knockout cells with the addition of C2 ceramide (right panel). C Western blot analysis of the expression of autophagy-related proteins (ATG5, Beclin1, and LC3II/LC3I). D Western blot analysis of phosphorylated mTOR, Beclin1, BCL2, and caspase3 in CRC cells with or without ECHS1 overexpression and Eliglustat (100 nmol). E Flow cytometry analysis of intracellular ROS content. Bars in the right panel represent the average fluorescence intensity. F IF assay analysis of JC-10 aggregates (red) and monomers (green) in the mitochondrial matrix with or without starvation (2 h) and Eliglustat (100 nmol). Bars of the right panel represent the ratio of the average fluorescence intensity of monomers (green) and aggregates (red). G Electron microscopy results show mitochondrial morphology (yellow arrows) and autophagosomes (red arrows) in ECHS1-knockout SW480 cells. H Histogram of flow cytometry assay showing the ratio of the average fluorescence intensity of apoptotic cells with or without Oxaliplatin (20 µg/µl) and Eliglustat (100 nmol).

Fig 4: Overexpression of ECHS1 is associated with tumor progression and poor prognosis of CRC.A Representative images of IHC staining analyses of LASP1 and ECHS1 in CRC tissues. B Western blot analysis of ECHS1 in CRC tissues (T) and adjacent nontumor tissues (N). The scatterplot on the right shows the relative expression of ECHS1 in normal and CRC tissues (normalized to GAPDH, P < 0.05). C Left panel: immunohistochemistry (IHC) analysis of ECHS1 in CRC tissues and adjacent nontumor tissues. Right panel: histogram representing IHC results in CRC tissues (T) and adjacent nontumor tissues (N) and with or without lymph-node metastasis. D Kaplan–Meier survival curves and univariate analyses (log rank) for CRC patients with distinct expression levels of ECHS1. E Sketch map of the mechanism of ECHS1-mediated CRC proliferation and drug resistance.

Fig 5: ECHS1 is essential for LASP1-mediated sphingomyelin-metabolism imbalance by interacting with SH3 domain of LASP1 in CRC.A Immunoprecipitation-assay analysis of endogenous interaction between ECHS1 and LASP1 in SW480 cells. B Western blot assay of LASP1 and ECHS1 protein and quantification of ECHS1 relative level at the indicated time in HCT116 cells transfected with siLASP1 or siNC (upper figure) or RKO cells transfected with LV-LASP1 or LV-Ctrl (lower figure) after CHX treatment to block protein synthesis. C Western blot assay of the level of LASP1 and ECHS1 protein with application of chloroquine (10uM) or MG132 (10uM) after 8 h. D Schematic presentation of LASP1 structure features. The structures of truncated LASP1 are listed behind those of the full-length proteins. E Western blotting results indicate the binding domain of ECHS1 and LASP1 in 293 T cells. F Western blot assay of HA and ECHS1 protein in 293 T cells transfected with plasmid HA-LASP1 full length (1–261aa), HA-LASP1 (60–199aa), or HA-LASP1(131–261aa) after CHX treatment to block protein synthesis. G Heatmap of the LC–MS assay for RKO-LASP1 and RKO-LASP1 transfected with siECHS1 cells. H Heatmap and histogram of depicting the differentially expressed lipomics for RKO-LASP1 and RKO-LASP1 plus siRNA-ECHS1 cells.