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: 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: Primary antibodies against METTL3 (15073-1-AP, 1:300; Proteintech, China) and TMBIM6 (26782-1-AP, 1:300; Proteintech, China) were

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: Primary antibodies against METTL3 (15073-1-AP, 1:300; Proteintech, China) and TMBIM6 (26782-1-AP, 1:300; Proteintech, China) were

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: Primary antibodies against METTL3 (15073-1-AP, 1:300; Proteintech, China) and TMBIM6 (26782-1-AP, 1:300; Proteintech, China) were

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: Primary antibodies against METTL3 (15073-1-AP, 1:300; Proteintech, China) and TMBIM6 (26782-1-AP, 1:300; Proteintech, China) were

Techniques: Expressing, Modification, Methylation

Journal: Progress in neuro-psychopharmacology & biological psychiatry

Article Title: MG53 attenuates lipopolysaccharide-induced neurotoxicity and neuroinflammation via inhibiting TLR4/NF-κB pathway in vitro and in vivo

doi: 10.1016/j.pnpbp.2019.109684

Figure Lengend Snippet: The effects of MG53 on cell cycle and cell apoptosis in LPS-induced HT22 cells. (A) Cell cycle was detected by flow cytometry. (B) Quantification of cell cycles. (C) Quantification of Annexin V/PI positive cells. (D) Apoptosis of HT22 was detected by Annexin V/PI staining. (E) Western blot and (F) densitometry analysis of Cyclin D1, Bax and Bcl-2. Data were presented as mean ± SEM. *p < .05, compared with NC group; # p < .05, compared with LPS group.

Article Snippet: The membrane was blocked in 5% skim milk and incubated with diluted primary antibodies: Cyclin Dl (1:500, Proteintech Group, Wuhan,China), Bcl-2 (1:200, Cell Signaling Technology, Danvers, MA, USA), Bax (1:200), TLR4 (1:500, CST), NF-κB (1:1000, CST), p-NF-κB (1:1000, CST), Iκβα (1:1000, CST), p –Iκβα (1: 1000, CST), IL-1β (1:500, Santa Cmz Biotechnology, Dallas, USA), IL-6 (1:500, Santa Cruz), TNF-α(1:500, Santa Cruz), β-actin (1:2000, Santa Cruz).

Techniques: Flow Cytometry, Staining, Western Blot

Journal: Progress in neuro-psychopharmacology & biological psychiatry

Article Title: MG53 attenuates lipopolysaccharide-induced neurotoxicity and neuroinflammation via inhibiting TLR4/NF-κB pathway in vitro and in vivo

doi: 10.1016/j.pnpbp.2019.109684

Figure Lengend Snippet: Effect of MG53 on neuronal cell death in LPS-treated mice. (A) Representative apoptotic neurons (red) in the hippocampus detected by TUNEL staining, Scale bar = 200 µm. (B) Quantitative analysis of TUNEL positive cells. (C) Representative immunoblots and (D) densitometric analysis of Bax and Bcl-2 in the hippocampus. β-actin was used as an internalcontrol. Data were presented as mean ± SEM. n = 6 per group. *p < .05, compared with Con group; # p < .05, compared with LPS group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The membrane was blocked in 5% skim milk and incubated with diluted primary antibodies: Cyclin Dl (1:500, Proteintech Group, Wuhan,China), Bcl-2 (1:200, Cell Signaling Technology, Danvers, MA, USA), Bax (1:200), TLR4 (1:500, CST), NF-κB (1:1000, CST), p-NF-κB (1:1000, CST), Iκβα (1:1000, CST), p –Iκβα (1: 1000, CST), IL-1β (1:500, Santa Cmz Biotechnology, Dallas, USA), IL-6 (1:500, Santa Cruz), TNF-α(1:500, Santa Cruz), β-actin (1:2000, Santa Cruz).

Techniques: TUNEL Assay, Staining, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: MicroRNA-26a-3p rescues depression-like behaviors in male rats via preventing hippocampal neuronal anomalies

doi: 10.1172/JCI148853

Figure Lengend Snippet: (A) Representative confocal microscopic images showing expression of cleaved caspase-3 and DCX within the DG. Scale bars: 50 μm. n = 6 rats per group and at least 4–6 images from 1 animal. (B) Overexpression of miR-26a-3p decreased protein levels of proapoptotic factors in CUMS rats. n = 6 rats per group. Western blotting results of Bcl-2, Bax, and caspase-9 were from the same samples and run in parallel in different gels. Independent biological replicate experiments were repeated 3 times. (C) Representative electron micrographs showing nuclear chromatin abnormalities in DG neurons. Scale bars: 1 μm. n = 4 rats per group and at least 20 micrographs from 1 animal. Immunofluorescence and electron microscopy experiments were repeated at least 3 times and quantitation was done for representative samples from each group. Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001 vs. WT; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. eGFP control (CUMS + AAV-eGFP) by 2-way ANOVA with Tukey’s post hoc correction.

Article Snippet: Proteins (30 μg) from each sample were electrophoretically resolved in 8%–15% SDS-PAGE gels, transferred to PVDF membranes, and probed with the following primary antibodies: anti-BDNF (1:300, catalog sc-546, Santa Cruz Biotechnology Inc.); anti-CREB (1:500, catalog 9197), anti–PSD-95 (1:1000, catalog 3450), anti-Syn (1:1000, catalog 5461), anti–LC3-I/LC3-II (1:1000, catalog 12741), anti–beclin-1 (1:1000, catalog 3495), anti-PARP (1:1000, catalog 9532), anti–cleaved caspase-3 (1:500, catalog 9661), anti-PTEN (1:500, catalog 9552), anti-PI3K (1:500, catalog 4292), anti–p-Akt (1:500, catalog 9271), anti-p53 (1:500, catalog 9284), anti-p62 (1:1000, catalog 23214), anti–β-actin (1:1000, catalog 4970) (all Cell Signaling Technologies); anti-caspase-9 (1:500, catalog AP0359, Bioworld); and anti-BAX (1:1000, catalog 50599-2-lg) and anti-GAPDH (1:4000, catalog 10494-1-AP) (both Proteintech Group).

Techniques: Expressing, Over Expression, Western Blot, Immunofluorescence, Electron Microscopy, Quantitation Assay

Journal: The Journal of Clinical Investigation

Article Title: MicroRNA-26a-3p rescues depression-like behaviors in male rats via preventing hippocampal neuronal anomalies

doi: 10.1172/JCI148853

Figure Lengend Snippet: (A) bpV(pic) treatment increased expression of PI3K and phosphorylated Akt and decreased p53 expression levels in miR-26a-3p–knockdown rats. Western blotting results of PI3K and p-Akt were from the same samples and run in parallel in different gels. n = 6 rats per group with 3 independent biological replicate experiments. (B) bpV(pic) treatment increased LC3-II/LC3-I and beclin-1 expression, and decreased expression of p62 in miR-26a-3p–knockdown rats. Western blotting results for p62 and beclin-1 were from the same samples and run in parallel in different gels. n = 6 rats per group with 3 independent biological replicate experiments. (C) bpV(pic) treatment increased protein levels of BDNF, PSD-95, and Syn within the DG of miR-26a-3p–knockdown rats. Western blotting results for Syn and BDNF were from the same samples and run in parallel in different gels. n = 6 per rats group with 3 independent biological replicate experiments. (D) bpV(pic) treatment decreased mRNA levels of Bax, caspase-3, and caspase-9, and increased Bcl-2 mRNA levels in miR-26a-3p–knockdown rats. n = 6 rats per group with 3 independent biological replicate experiments. (E) bpV(pic) treatment in miR-26a-3p–knockdown rats increased sucrose consumption in the sucrose preference test and (F) decreased immobility times and increased swimming times in the forced-swim test. (G) bpV(pic) treatment in DG neurons produced changes in spontaneous burst activity. n = 16 rats per group. Each data point represents 1 animal. Electrophysiological recordings were repeated at least 3 times. Data are presented as mean ± SEM. n = 18 rats per group in behavioral tests. **P < 0.01, ***P < 0.001 vs. WT; ##P < 0.01, ###P < 0.001 vs. AAV-26a-sponge (WT + AAV-miR-26a-sponge) by 2-way ANOVA with Tukey’s post hoc correction.

Article Snippet: Proteins (30 μg) from each sample were electrophoretically resolved in 8%–15% SDS-PAGE gels, transferred to PVDF membranes, and probed with the following primary antibodies: anti-BDNF (1:300, catalog sc-546, Santa Cruz Biotechnology Inc.); anti-CREB (1:500, catalog 9197), anti–PSD-95 (1:1000, catalog 3450), anti-Syn (1:1000, catalog 5461), anti–LC3-I/LC3-II (1:1000, catalog 12741), anti–beclin-1 (1:1000, catalog 3495), anti-PARP (1:1000, catalog 9532), anti–cleaved caspase-3 (1:500, catalog 9661), anti-PTEN (1:500, catalog 9552), anti-PI3K (1:500, catalog 4292), anti–p-Akt (1:500, catalog 9271), anti-p53 (1:500, catalog 9284), anti-p62 (1:1000, catalog 23214), anti–β-actin (1:1000, catalog 4970) (all Cell Signaling Technologies); anti-caspase-9 (1:500, catalog AP0359, Bioworld); and anti-BAX (1:1000, catalog 50599-2-lg) and anti-GAPDH (1:4000, catalog 10494-1-AP) (both Proteintech Group).

Techniques: Expressing, Western Blot, Produced, Activity Assay