Fig 1: CERS1 expression attenuates the PI3K/AKT/mTOR signaling pathway. (A) Expression of indicated proteins was evaluated using western blotting. (B,C) The effect of LY294002 treatment on the proliferation, migration, and invasion abilities of NSCLC cells stably transfected with shCERS1 was tested via wound-healing and transwell assays. (D) After adding LY294002, the ability of CERS1 knockdown cells to cross the BBB was assessed. * p < 0.05; ** p < 0.01; *** p < 0.001.
Fig 2: CERS1 overexpression inhibits the proliferation, migration, and invasion of NSCLC cells. (A,B) The efficiency of CERS1 overexpression and depletion was analyzed using RT-qPCR and western blotting. (C) The ability of H1299 and PC-9 cells to proliferate following CERS1 transfection was tested using CCK-8 assays. (D) MMP-9 expression was evaluated using western blotting. (E) The ability of H1299 and PC-9 cells to migrate and invade was tested through transwell assays. (F) KEGG and GSEA analyses were used to determine the signaling pathway associated with CERS1. (G) The impact of CERS1 on cell apoptosis was detected using western blotting. (H) Effect of CERS1 on the ability of lung cancer cells to penetrate the BBB model. * p < 0.05; ** p < 0.01; *** p < 0.001.
Fig 3: Interaction between CERS1 and transcription factor USP14. (A) Expression of indicated proteins was evaluated using western blotting. (A) USP14 expression following CERS1 knockdown was detected using western blotting. (B) Co-IP demonstrated an interaction between CERS1 and USP14. (C) Immunofluorescence was performed to detect CERS1 and USP14 localization in cells.
Fig 4: CERS1 exhibits low expression in patients with NSCLC BM. (A) mRNA levels of CERS1-6 in fresh NSCLC and BM tissues. (B) mRNA expression of CERS1-6 in NSCLC cell lines with different BM tendencies. (C) CERS1 expression in lung adenocarcinoma and normal tissues based on TCGA and GTEx database analyses. (D) CERS1 expression in NSCLC cell lines was tested via western blotting. (E) Representative immunohistochemical staining images of CERS1 in lung tissues without BM (BM−), lung tissue of BM+, and brain tissue of BM+. (F) A summary of immunohistochemical staining results for CERS1 expression. (G) Kaplan–Meier plotted the overall survival of patients stratified by CERS1 expression. ns, no-significant; * p < 0.05; ** p < 0.01; *** p < 0.001.
Fig 5: CERS1 suppresses tumorigenesis and brain metastasis in vivo. (A) PC-9 cells with stable knockdown and overexpression of CERS1 were used to establish a subcutaneous xenograft model, and the tumor growth curves were plotted. (B) Expression of CERS1 and MMP9 in subcutaneously transplanted tumors was detected through immunohistochemical staining. (C) Bioluminescent images of mice injected with CERS1-overexpressing cells in the orthotopic xenograft model. (D) Hematoxylin and eosin-stained images (40× and 200×) of mice with brain metastases. ** p < 0.01; *** p < 0.001.
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