Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: ( A ) SH3 domain GRB2-like endophilin B1 (SH3GLB1) and superoxide dismutase 2 (SOD2) levels were simultaneously enhanced in the patient derived xenografts (PDX) model of naïve glioblastoma (GBM) tumors after temozolomide (TMZ) (5 mg/kg) treatment for three weeks. ( B ) The results show increased levels of 2 ′ ,7 ′ -dichlorodihydrofluorescein diacetate (H 2 DCFDA) staining (a reactive oxygen species (ROS) detection probe) in parental U87MG (for 6 h) or A172 (for 24 h) cells after triple co-incubation with TMZ, ATZ, and HNE. H 2 O 2 : 100 μM. Scale bar: 50 μm

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Derivative Assay, Staining, Incubation

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: SH3 domain GRB2-like endophilin B1 (SH3GLB1) is a downstream protein of superoxide dismutase 2 (SOD2). ( A ) The biological network of SH3GLB1-mediated autophagy and SOD2-involved antioxidants was predicted using the STRING bioinformatics tool. ( B ) Resistant cells were transfected with the microtubule-associated protein 1 light chain 3-Enhanced Green Fluorescent Protein (LC3-EGFP) plasmid, and representative fluorescent images for the formation of LC3-EGFP dots (puncta) are shown. Scale bar: 4 μm. ( C ) Protein immunoblotting showing that the resistant cells treated with temozolomide (TMZ) for 24 h induced autophagic reactions, which were attenuated by pretreatment with diethyldithiocarbamic acid (DETC; an SOD inhibitor). ( D ) Immunohistochemical (IHC) staining demonstrating SH3GLB1 expression in primary, recurrent glioblastoma (GBM-R) cells inoculated subcutaneously into mice receiving the indicated treatments for 15 days. A statistical graph is shown in the right-hand panel. Scale bar: 200 μm. ( E ) SOD2 siRNA or ( F ) overexpression vectors were used in U87MG- and A172-resistance cells or U87MG- and A172-parental cells, respectively, three days after transfection to examine the association between SOD2 and SH3GLB1. ( G ) SH3GLB1 siRNA was used in U87MG- and A172-resistance cells, and the association between SH3GLB1 and SOD2 was studied using western blotting. For each blot, the adjacent bar chart depicts the fold change relative to control. N = 3 in each group. * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Transfection, Plasmid Preparation, Western Blot, Immunohistochemical staining, Immunohistochemistry, Expressing, Over Expression, Control

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: SH3GLB1 levels are regulated by hydrogen peroxide. ( A ) Blots show that co-treatment with TMZ + 4-hydroxynonenal (HNE) + 3-amino-1,2,4-triazole (ATZ), enhanced the expression of SH3GLB1 in parental cells. ( B ) Cells were pretreated with N-acetyl-L-cysteine (NAC) and subjected to three co-treatments. U87MG cells were co-treated for 8 h and A172 cells for 18 h. Intracellular H 2 O 2 levels were measured, and ( C ) SH3GLB1, p-AKT (Ser473), and SOD2 levels were detected by western blotting. ( D ) MK-2206 inhibits p-Akt (Ser473) expression. ( E ) Blots showing the levels of the indicated proteins after co-treatment with TMZ, SC-79 (2-Amino-6-chloro-α-cyano-3-(ethoxycarbonyl)-4H-1-benzopyran-4-acetic acid ethyl ester), and NAC. TMZ: 100 μM, ATZ: 20 mM, HNE: 10 μM, NAC: 10 mM, MK-2206: 5 μ, SC-79: 10 μg/mL. For each blot, the adjacent bar chart depicts the fold change relative to control. N = 3 in each group. * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Expressing, Western Blot, Control

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: Increased levels of hydrogen peroxide demonstrate different effects on SH3GLB1 expression in the resistant cells. ( A ) MK-2206 was pretreated in the TMZ-treated resistant cells. ( B ) IHC staining demonstrating p-AKT levels in U87MG-R cells transfected with shSH3GLB1 or shControl vectors and inoculated subcutaneously into mice receiving the indicated treatments for 23 days. A statistical graph is shown in the right-hand panel. The arrows indicate the positive staining of SH3GLB1. Scale bar: 200 μm. ( C ) The resistant cells were treated with the indicated reagents. U87MG-R cells were co-treated for 18 h and A172-R cells for 8 h. The levels of intracellular H 2 O 2 were measured. ( D ) Protein immunoblotting after stimulation and rescue treatments. ( E ) Resistant cells were co-treated with TMZ and NAC. TMZ: 100 μM, MK-2206: 5 μM, ATZ: 20 mM, HNE: 10 μM, NAC: 10 mM, MK-2206: 5 μM. For each blot, the adjacent bar chart depicts the fold change relative to control. N = 3 in each group. * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Expressing, Immunohistochemistry, Transfection, Staining, Western Blot, Control

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: ( A ) Blots showing the levels of the indicated proteins. Resistant cells were treated with TMZ with or without MK-2206. TMZ: 100 μM, MK-2206: 5 μM ( B ) The parental and resistant cells are treated with increasing concentrations of extracellular H 2 O 2 for 24 h. H 2 O 2 concentrations are indicated. The summary graph demonstrates the difference in the expression of SH3GLB1 in response to extracellular H 2 O 2 between the two cell lines. ** p < 0.01 and *** p < 0.001

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Expressing

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: TMZ combined with the inhibitors of an H 2 O 2 -related enzyme affects autophagy levels and tumor growth in the resistant cells. ( A ) The triple co-treatment caused simultaneous changes in autophagy and SH3GLB1 expression. ( B ) Proliferation assay results for parental or resistant cells treated with the indicated compounds are shown as bar graphs, suggesting that the resistant cells were more susceptible to H 2 O 2 accumulation. ( C ) Morphology of A172 and A172-R cells after 72 h of treatment. Control group is no TMZ-treated group. Scale bar: 100 μm. ( D ) Arrows indicate the inhibition of SH3GLB1 levels in the TMZ+HNE and TMZ+HNE+ATZ groups. ( E ) The mice were subcutaneously injected with luciferase-expressing U87MG-R cells. Bioluminescence signals were recorded on the indicated days using an IVIS imaging system. The growth curves of the tumors were analyzed according to the bioluminescence intensity. (N = 5 in each group) ( F ) Immunoblots showing the protein levels of xenograft tumor lysates from mice harvested after the indicated treatments. TMZ: 5 mg/kg, HNE: 2.5 mg/kg ( G ) The IHC staining demonstrates autophagy levels in shSH3GLB1 or shControl group of U87MG-R cells subcutaneously injected in mice and those receiving the indicated treatments. The statistic graph is shown in the right panel. Scale bar: 200 μm. Scale bar in the enlarged graph represents 1 mm. For each blot, the adjacent bar chart depicts the fold change relative to control. N = 3~5 in each group. * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Expressing, Proliferation Assay, Control, Inhibition, Injection, Luciferase, Imaging, Western Blot, Immunohistochemistry

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: Mitochondrial dysfunction affects SH3GLB1 expression via H 2 O 2 /AKT signaling. ( A ) H 2 O 2 levels in resistant cells were measured after the indicated treatments. TMZ: 100 μM, CCCP: 10 μM ( B ) The protein immunoblot shows that SH3GLB1 levels are regulated by treating with CCCP for 24 h with or without TMZ. Resistant cells (U87MG-R) were treated with TMZ with or without HgCl 2 . ( C ) The statistic graph shows H 2 O 2 levels in the indicated treatments. ( D ) The blots demonstrate the indicated protein expression after the treatments. The statistic graphs are shown. TMZ: 100 μM, HgCl 2 : 20 μM. For each blot, the adjacent bar chart depicts the fold change relative to control. N = 3 in each group. * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Expressing, Western Blot, Control

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: ( A ) SH3GLB1 and Bax levels were compared between normal and GBM tissues from the TCGA-GBM dataset. ( B ) The protein immunoblots showed levels of Bax-α (21 kd; the lower arrow) and Bax-β (24 kd; the upper arrow) in the parental cells and the derived resistant cells. Corresponding fold-change values (relative to control) are shown in the lower box. ( C ) The western blotting showed that the resistant cells (A172-R) were treated with TMZ with or without HgCl 2 . TMZ: 100 μM, HgCl 2 : 20 μM. Corresponding fold-change values (relative to control) are shown beneath the Western blot panels. N = 3 in each group

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Western Blot, Derivative Assay, Control

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: TMZ elevates reactive oxygen species (ROS) levels, including H 2 O 2 . In resistant GBM cells, moderate H 2 O 2 activates AKT, driving SH3GLB1 expression and sustaining drug resistance, whereas excessive H 2 O 2 (e.g., following HNE co-treatment) suppresses SH3GLB1 and impairs resistance. Furthermore, HgCl 2 downregulates mitochondrial aquaporin-9 (AQP9), reducing H 2 O 2 flux and SH3GLB1 expression. This schematic illustrates how H 2 O 2 levels, AKT activation, and AQP9 modulation collectively shape SH3GLB1-driven resistance

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Expressing, Activation Assay