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

Journal: Drug Design, Development and Therapy

Article Title: Obacunone Promotes Functional Recovery After Spinal Cord Injury by Attenuating Neuroinflammation by Targeting the TLR4/MyD88/p38 MAPK Pathway

doi: 10.2147/DDDT.S577707

Figure Lengend Snippet: Oba attenuated neuroinflammation and reduced cellular apoptosis following SCI. All experiments were performed using tissues from the Sham, SCI, and SCI + Oba (20 mg/kg) groups at 3 days post-SCI. ( A ) Representative Western blot image of iNOS, IL-1β and TNFα at 3 days post-SCI. ( B ) Quantitative analysis of iNOS, IL-1β and TNFα protein levels (n = 5). ( C ) Representative immunofluorescence images of iNOS (green) and DAPI (blue) in spinal cord tissue at 3 days post-SCI; Scale bar = 50 μm. ( D ) Quantitative analysis of iNOS fluorescence areas (n = 5). ( E ) Representative Western blot image of Bax and Cleaved-caspase3 at 3 days post-SCI. ( F ) Quantitative analysis of Bax and Cleaved-caspase3 protein levels (n = 5). ( G ) Representative immunofluorescence images of TUNEL (red) and DAPI (blue) in spinal cord tissue at 3 days post-SCI; Scale bar = 50 μm. ( H ) Quantitative analysis of TUNEL positive cells (n = 5). The values are presented as mean ± SD. *p<0.05, **p<0.01, ***p<0.001; NS, not significant.

Article Snippet: The primary antibodies included rabbit anti-iNOS (13120, Cell signaling technology, 1: 1000), rabbit anti-TNFα (IPB9396, Baijia, 1:1000), rabbit anti-IL-1β (IPB0002, Baijia, 1: 1000), rabbit anti-GAP43 (16971-1-AP, Proteintech, 1:1000), mouse anti- α-tubulin (11224-1-AP, Proteintech, 1:1000), mouse anti-GAPDH (ab307799, Abcam, 1:5000), rabbit anti-Cleaved-caspase3 (AF7022, Affinity, 1: 1000), rabbit anti-Bax (505992-Ig, Proteintech, 1: 1000), rabbit anti-TLR4 (A17436, ABclonal, 1: 1000), rabbit anti-p38 MAPK ( T55600 , ABmart, 1: 1000), rabbit anti-Phospho-p38 MAPK (Thr180/Tyr182) (TA4001, ABmart, 1: 1000), rabbit anti-MyD88 (YM8747, immunoway, 1: 1000).

Techniques: Western Blot, Immunofluorescence, Fluorescence, TUNEL Assay

Journal: Drug Design, Development and Therapy

Article Title: Obacunone Promotes Functional Recovery After Spinal Cord Injury by Attenuating Neuroinflammation by Targeting the TLR4/MyD88/p38 MAPK Pathway

doi: 10.2147/DDDT.S577707

Figure Lengend Snippet: Oba suppresses microglial inflammation and blocks the subsequent apoptosis of HT22 neurons. ( A ) Viability of BV-2 cells treated with different concentrations of Oba, as assessed by CCK-8 assay (n = 5). ( B ) Representative Calcein AM (green)/PI (red) staining images of BV-2 cells following treatment with Oba at the indicated concentrations (0, 25, and 50 μM). Scale bar = 100 μm. ( C ) Quantitative analysis of the ratio of viable cells (n = 5). ( D ) Representative Western blot images of iNOS, TNFα, and IL-1β expression in BV-2 cells. ( E ) Quantitative analysis of iNOS, TNFα and IL-1β protein levels (n = 5). ( F ) Representative immunofluorescence images of iNOS (green) and DAPI (blue) in BV-2 cells treated as follows: vehicle (Control), LPS, LPS+Oba 25 μM, and LPS+Oba 50 μM. Scale bar = 50 μm. ( G ) Quantitative analysis of fluorescence intensity of iNOS (n = 5). ( H ) mRNA expression levels of TNFα and IL-1β in BV-2 cells measured by qPCR (n = 6). ( I ) Representative Western blot image of Bax and Cleaved-caspase3 protein level in HT22 cells. ( J ) Quantitative analysis of Bax and Cleaved-caspase3 protein levels (n = 5). ( K ) Representative flow cytometry plots of HT22 cells stained with Annexin V-FITC (x-axis) and PI (y-axis). ( L ) The percentage of apoptotic HT22 cells, as determined by flow cytometry. (n = 5). The plus and minus signs (+, -) denote the presence or absence of the following reagents in culture: LPS, 25 μM Oba, and 50 μM Oba. The values are presented as mean ± SD. *p<0.05, **p<0.01, ***p<0.001; NS, not significant.

Article Snippet: The primary antibodies included rabbit anti-iNOS (13120, Cell signaling technology, 1: 1000), rabbit anti-TNFα (IPB9396, Baijia, 1:1000), rabbit anti-IL-1β (IPB0002, Baijia, 1: 1000), rabbit anti-GAP43 (16971-1-AP, Proteintech, 1:1000), mouse anti- α-tubulin (11224-1-AP, Proteintech, 1:1000), mouse anti-GAPDH (ab307799, Abcam, 1:5000), rabbit anti-Cleaved-caspase3 (AF7022, Affinity, 1: 1000), rabbit anti-Bax (505992-Ig, Proteintech, 1: 1000), rabbit anti-TLR4 (A17436, ABclonal, 1: 1000), rabbit anti-p38 MAPK ( T55600 , ABmart, 1: 1000), rabbit anti-Phospho-p38 MAPK (Thr180/Tyr182) (TA4001, ABmart, 1: 1000), rabbit anti-MyD88 (YM8747, immunoway, 1: 1000).

Techniques: CCK-8 Assay, Staining, Western Blot, Expressing, Immunofluorescence, Control, Fluorescence, Flow Cytometry

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Methyltransferase-like 3 aggravates endoplasmic reticulum stress in preeclampsia by targeting TMBIM6 in YTHDF2-dependent manner.

doi: 10.1186/s10020-023-00604-x

Figure Lengend Snippet: Fig. 5 Attenuated TG-induced ER stress, oxidative stress and apoptosis in HTR-8/SVneo cells by overexpressing TMBIM6. A–H Representative WB images and densitometry quantification of TMBIM6, GRP78, CHOP, NRF2, HO-1, Bax and Bcl-2 in different groups. The data were normalized to β-actin. I, J ROS production detected by DCFH-DA. Photographs were obtained at 100 × (Scale bar 100 μm). K, L Apoptosis rate in different groups detected by flow cytometry. M Representative transwell photos of HTR-8/SVneo cells in different groups (100 × magnification, Scale bar 100 μm). N Qualified data shown in M. Oe-Con: Overexpression of control plasmid; Oe-TMBIM6: Overexpression of TMBIM6 plasmid. n = 3. *p < 0.05, **p < 0.01

Article Snippet: Equal amounts of proteins from different groups were separated by 10% SDS-PAGE gels and transferred onto PVDF membranes (0.45 mm pore size; Millipore, USA) which were then blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h. Next, the membranes were incubated with the METTL3 (15073-1-AP, 1:1000; Proteintech, China), TMBIM6 (26782-1-AP, 1:1000; Proteintech, China), GRP78 (11587-1-AP, 1:1000; Proteintech, China), CHOP (2895T, 1:1000; Cell Signaling Technology, USA), METTL14 (26,158–1-AP, 1:1000; Proteintech, China), fat mass and obesity-associated protein (FTO; 27226-1-AP, 1:1000; Proteintech, China), B-cell lymphoma-2 (Bcl-2; 26593-1-AP, 1:1500; Proteintech, China), Bcl-2-associated X (Bax; 2772T, 1:1000; Cell Signaling Technology, USA), heme oxygenase-1 (HO-1; 10701-1-AP, 1:3000; Proteintech, China), nuclear factor erythroid 2-related factor 2 (NRF2; 16396-1-AP; 1:1000; Proteintech, China), YTHDF2 (24744-1-AP, 1:4000; Proteintech, China), and β-actin (66009-1-Ig, 1:10,000; Proteintech, China) antibodies overnight at 4 °C.

Techniques: Flow Cytometry, Over Expression, Control, Plasmid Preparation

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Methyltransferase-like 3 aggravates endoplasmic reticulum stress in preeclampsia by targeting TMBIM6 in YTHDF2-dependent manner.

doi: 10.1186/s10020-023-00604-x

Figure Lengend Snippet: Fig. 6 Overexpression of TMBIM6 partially neutralized the effects of METTL3 on TG-induced ER stress, oxidative stress and apoptosis in HTR-8/SVneo cells. A–I Representative WB images and densitometry quantification of METTL3, TMBIM6, GRP78, CHOP, NRF2, HO-1, Bax and Bcl-2 in different groups. The data were normalized to β-actin. J, K ROS production detected by DCFH-DA. Photographs were obtained at 100 × (Scale bar 100 μm). L, M Apoptosis rate in different groups detected by flow cytometry. N Representative transwell photos of HTR-8/SVneo cells in different groups (100 × magnification, Scale bar 100 μm). O Qualified data shown in L. Oe-Con: Overexpression of control plasmid; Oe-METTL3: Overexpression of METTL3 plasmid; Oe-TMBIM6: Overexpression of TMBIM6 plasmid. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Equal amounts of proteins from different groups were separated by 10% SDS-PAGE gels and transferred onto PVDF membranes (0.45 mm pore size; Millipore, USA) which were then blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h. Next, the membranes were incubated with the METTL3 (15073-1-AP, 1:1000; Proteintech, China), TMBIM6 (26782-1-AP, 1:1000; Proteintech, China), GRP78 (11587-1-AP, 1:1000; Proteintech, China), CHOP (2895T, 1:1000; Cell Signaling Technology, USA), METTL14 (26,158–1-AP, 1:1000; Proteintech, China), fat mass and obesity-associated protein (FTO; 27226-1-AP, 1:1000; Proteintech, China), B-cell lymphoma-2 (Bcl-2; 26593-1-AP, 1:1500; Proteintech, China), Bcl-2-associated X (Bax; 2772T, 1:1000; Cell Signaling Technology, USA), heme oxygenase-1 (HO-1; 10701-1-AP, 1:3000; Proteintech, China), nuclear factor erythroid 2-related factor 2 (NRF2; 16396-1-AP; 1:1000; Proteintech, China), YTHDF2 (24744-1-AP, 1:4000; Proteintech, China), and β-actin (66009-1-Ig, 1:10,000; Proteintech, China) antibodies overnight at 4 °C.

Techniques: Over Expression, Flow Cytometry, Control, Plasmid Preparation

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Methyltransferase-like 3 aggravates endoplasmic reticulum stress in preeclampsia by targeting TMBIM6 in YTHDF2-dependent manner.

doi: 10.1186/s10020-023-00604-x

Figure Lengend Snippet: Fig. 7 METTL3 affected the expression of TMBIM6 by regulating TMBIM6 mRNA stability via YTHDF2 involvement. A–C The knockdown efficiency of different sh-YTHDF2s determined by qRT-PCR and WB. D The relative expression levels of the TMBIM6 mRNA in sh-Con and sh-YTHDF2 group determined by qRT-PCR. E–H Representative WB images and densitometry quantification of TMBIM6 in different groups. The data were normalized to β-actin. I, J Transcript stability assay of TMBIM6 in different groups. n = 3. ns: no significance, *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Equal amounts of proteins from different groups were separated by 10% SDS-PAGE gels and transferred onto PVDF membranes (0.45 mm pore size; Millipore, USA) which were then blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h. Next, the membranes were incubated with the METTL3 (15073-1-AP, 1:1000; Proteintech, China), TMBIM6 (26782-1-AP, 1:1000; Proteintech, China), GRP78 (11587-1-AP, 1:1000; Proteintech, China), CHOP (2895T, 1:1000; Cell Signaling Technology, USA), METTL14 (26,158–1-AP, 1:1000; Proteintech, China), fat mass and obesity-associated protein (FTO; 27226-1-AP, 1:1000; Proteintech, China), B-cell lymphoma-2 (Bcl-2; 26593-1-AP, 1:1500; Proteintech, China), Bcl-2-associated X (Bax; 2772T, 1:1000; Cell Signaling Technology, USA), heme oxygenase-1 (HO-1; 10701-1-AP, 1:3000; Proteintech, China), nuclear factor erythroid 2-related factor 2 (NRF2; 16396-1-AP; 1:1000; Proteintech, China), YTHDF2 (24744-1-AP, 1:4000; Proteintech, China), and β-actin (66009-1-Ig, 1:10,000; Proteintech, China) antibodies overnight at 4 °C.

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Stability Assay

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Methyltransferase-like 3 aggravates endoplasmic reticulum stress in preeclampsia by targeting TMBIM6 in YTHDF2-dependent manner.

doi: 10.1186/s10020-023-00604-x

Figure Lengend Snippet: Fig. 9 The schematic of molecular mechanism about how METTL3 affected TMBIM6 expression resulting in trophoblastic dysfunction and PE. ① When placentas got injured by many detrimental factors, such as I/R, the trophoblasts were under ER stress and METTL3 expression was upregulated increasing the m6A modification of TMBIM6 mRNA in trophoblasts. ② The methylated TMBIM6 mRNA was recognized by YTHDF2. ③ YTHDF2 decreased the stability of TMBIM6 mRNA and increased its degradation. ④ TMBIM6 expression was downregulated due to the decreased TMBIM6 mRNA and its translation. ⑤ Downregulation of TMBIM6 could not maintain the ER homeostasis, which aggravated ER stress. Moreover, the apoptosis and ROS production were increased when TMBIM6 was decreased. ⑥ The trophoblasts functions were impaired with the placental functions abnormal, which consequently contributed to PE

Article Snippet: Equal amounts of proteins from different groups were separated by 10% SDS-PAGE gels and transferred onto PVDF membranes (0.45 mm pore size; Millipore, USA) which were then blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h. Next, the membranes were incubated with the METTL3 (15073-1-AP, 1:1000; Proteintech, China), TMBIM6 (26782-1-AP, 1:1000; Proteintech, China), GRP78 (11587-1-AP, 1:1000; Proteintech, China), CHOP (2895T, 1:1000; Cell Signaling Technology, USA), METTL14 (26,158–1-AP, 1:1000; Proteintech, China), fat mass and obesity-associated protein (FTO; 27226-1-AP, 1:1000; Proteintech, China), B-cell lymphoma-2 (Bcl-2; 26593-1-AP, 1:1500; Proteintech, China), Bcl-2-associated X (Bax; 2772T, 1:1000; Cell Signaling Technology, USA), heme oxygenase-1 (HO-1; 10701-1-AP, 1:3000; Proteintech, China), nuclear factor erythroid 2-related factor 2 (NRF2; 16396-1-AP; 1:1000; Proteintech, China), YTHDF2 (24744-1-AP, 1:4000; Proteintech, China), and β-actin (66009-1-Ig, 1:10,000; Proteintech, China) antibodies overnight at 4 °C.

Techniques: Expressing, Modification, Methylation