Journal: Cell Death & Disease

Article Title: Autophagy-dependent secretion of ENO1 mediates chemoresistance of glioblastoma and tumor microenvironment remodeling

doi: 10.1038/s41419-025-08313-5

Figure Lengend Snippet: A Secretome profiling via 4D-FastDIA proteomics and GeneCards database screening identified secreted proteins. B Heatmap analysis of the identified secreted proteins. C Immunofluorescence (IF) staining demonstrating ENO1 colocalization with autophagy marker LC3B. Scale bars, 20 μm. D Western blot and ELISA analysis of secreted ENO1 levels in conditioned media from GBM cell lines exposed to TMZ (0–1000 μM, 48 h). E Time-course analysis (fixed 600 μM TMZ) monitoring ENO1 secretion by Western blot and ELISA. F Extracellular ENO1 quantification by ELISA and membrane integrity assessment via LDH release assay in 600 μM TMZ-treated models. G Co-immunoprecipitation (Co-IP) of GBM cell lysates using anti-ENO1 antibody, followed by immunoblotting with anti-TRIM16 and anti-SEC22B antibodies. H Western blot analysis of GAL3 and GAL8 expression in GBM cells treated with 600 μM TMZ. I IF double-labeling showing LLOMe (0.5 μM)-induced subcellular colocalization of LC3B and TRIM16. Scale bars, 20 μm. J Dose-response analysis: GBM cells treated with LLOMe (0, 0.25, 0.5, 1 μM; 24 h). Intracellular ENO1, LC3B-I-to-II conversion (Western blot), and secreted ENO1 (ELISA) were concurrently measured. K Time-course analysis (fixed 1 μM LLOMe; 0, 6, 12, 24 h): Intracellular ENO1/LC3B (Western blot) and secreted ENO1 (ELISA). L ATG5-knockdown validation: shNC/shATG5-transfected GBM cells ± TMZ (600 μM, 24 h). Immunoblotting for ATG5, secreted ENO1, and LC3B lipidation; secreted ENO1 quantified by ELISA. M Bafilomycin A1 (BafA1, 200 nM, 6 h pre-treatment) ± TMZ (600 μM, 24 h). Immunoblotting of whole-cell lysates (WCL) and conditioned media (CM) for LC3B/ENO1; secreted ENO1 by ELISA. N 3-Methyladenine (3-MA, 5 mM, 6 h pre-treatment) ± TMZ (600 μM, 24 h). Immunoblotting of WCL and CM for LC3B/ENO1; secreted ENO1 by ELISA. Data are expressed as mean ± SEM. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Antibodies used for target protein IP included: ENO1 Rabbit pAb (Proteintech, Cat# 11204-1-AP; 1:100 dilution), SEC22B Rabbit pAb (Proteintech, Cat# 14776-1-AP; 1:100 dilution), TRIM16 Rabbit pAb (Proteintech, Cat# 24403-1-AP; 1:100 dilution), TLR4 Mouse mAb (Abcam, Cat# ab22048; 1:100 dilution) and control IgG (Proteintech, Cat# 30000-0-AP; 1:100 dilution).

Techniques: Immunofluorescence, Staining, Marker, Western Blot, Enzyme-linked Immunosorbent Assay, Membrane, Lactate Dehydrogenase Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Labeling, Knockdown, Biomarker Discovery, Transfection