Fig 2: AEG-1 overexpression enhances TGF-ß1-induced autophagy and EMT in malignant glioma cells(A) Transmission electron microscopy images of U87 malignant glioma cells after transfection with pcDNA3.1-AEG-1/pcDNA3.1. N, nuclear; ER, endoplasmic reticulum; M, mitochondrion; AP, initial autophagic phagophore (isolation membrane); AV1, autophagosomes as autophagic vacuoles 1; AV2, autolysosomes as autophagic vacuoles 2. Scale bars: 500 nm (upper) and 200 nm (lower). (B) U251 and U87 cells are transfected with pcDNA3.1-GFP-MAP-LC3 and pcDNA3.1-AEG-1/pcDNA3.1 using Lipofectamine™ 2000 for 24 hours and then treated with TGF-ß1 (5 ng/ml) or vehicle for another 24 hours. GFP-MAP-LC3-labeled cells are monitored by fluorescence microscopy at 600 × magnification. Scale bar: 200 µm. (C) Quantification of the percentage of cells with focal GFP-MAP-LC3 at the indicated times after treatment. Error bars correspond to SD of three independent experiments (200 cells were counted per experiment). **, ##P < 0.01 compared to vehicle-treated cells. $$, &&P < 0.01 compared to pcDNA3.1-transfected cells. (D and E) U251 and U87 cells are transfected with pcDNA3.1-AEG-1/pcDNA3.1 using Lipofectamine™ 2000 for 24 hours, the expression of AEG-1 and cyclin D1 and MAP-LC3 lipidation and BECN1, E-cadherin, and vimentin are analyzed by western blot. ß-actin is used as internal controls to ascertain equal loading. (F and H) U251 and U87 cells are transfected with pcDNA3.1-AEG-1/pcDNA3.1 using Lipofectamine™ 2000 for 24 hours and then treated with TGF-ß1 (5 ng/ml) or vehicle for another 24 hours. The expression of AEG-1, MAP-LC3 lipidation and BECN1, E-cadherin, vimentin, Snail, Slug and Twist are analyzed by western blot. ß-actin is used as internal controls to ascertain equal loading. (G and I) Representative quantitative data of densitometric analyses. Data represent the mean ± SD of three different experiments. *P < 0.05 and **P < 0.01 compared to vehicle-treated cells or si Control-transfected cells.

Fig 3: Autophagy inhibition rescued apoptosis in human peritoneal mesothelial cells. (A and B) Human peritoneal mesothelial cells pre-treated with TGF-ß1 (10 ng/ml) or transfected with siRNA Beclin1 were treated with 4.25% high-glucose peritoneal dialysis solution (HGPDS) or vehicle for 24 hrs. The sum of early and late apoptotic cells ratio (%) was quantitated by flowcytometer analysis of Annexin V/PI. Results from three independent experiments are shown as means ± S.D. Results from three independent experiments are shown as means ± S.D. **P < 0.01, *P < 0.05 versus untreated control group. ## P < 0.01, # P < 0.05 versus 4.25% HGPDS-treated group. (C and D) Human peritoneal mesothelial cells transfected with siRNA Beclin1 were treated with 4.25% HGPDS or vehicle for 24 hrs. The apoptosis-related protein such as Bax, Bcl-2, Mcl-1, PARP, caspase-3 and caspase-8 was assessed by Western blotting assay. GAPDH was used as loading control.

Fig 4: The expression of AEG-1, autophagy hallmarks and EMT is high in diverse malignant glioma tumors(A) Representative photomicrographs of TGF-ß1, AEG-1, MAP-LC3, BECN1, N-cadherin and E-cadherin expression as determined by IHC staining in 6 samples of anaplastic astrocytoma, 6 samples of oligodendroglioma and oligoastrocytoma, 6 samples of glioblastoma multiforme (GBM), and 6 samples of morphologically normal brain tissue. NB, normal brain; A, anaplastic astrocytoma (grade II); O, oligodendroglioma and oligoastrocytoma (grade III); GBM, glioblastoma multiforme (grade IV). Image magnification: 200×. (B) Density mean of TGF-ß1, AEG-1, MAP-LC3, BECN1, N-cadherin and E-cadherin expression of diverse malignant gliomas determined by Image Pro-Plus. (C) Log10IOD of TGF-ß1, AEG-1, MAP-LC3, BECN1, N-cadherin and E-cadherin expression of diverse malignant gliomas determined by Image Pro-Plus. IOD indicates integrated optical density.

Fig 5: BECN1 silencing relieves sorafenib-triggered ferroptosis in HCC cells: (A) Silencing of BECN1 by siRNA in Huh7 cells alleviated sorafenib-induced ferroptotic cell death, (B) restored system Xc-’s transporter activity, (C) replenished intracellular total GSH, (D) reduced lipid ROS levels, and (E) restrained lipid peroxidation. Columns: mean; bars: SD (n = 3). Statistical significance was evaluated using the Mann–Whitney U test (*, p < 0.05; **, p < 0.01).