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: 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: Oncology Reports

Article Title: Downregulation of RNF138 inhibits cellular proliferation, migration, invasion and EMT in glioma cells via suppression of the Erk signaling pathway

doi: 10.3892/or.2018.6744

Figure Lengend Snippet: Apoptotic rates of U87 and U251 transduced with RNF138-siRNA and negative control lentivirus. (A) Transfection with RNF138-siRNA in U87 and U251 cells significantly increased cell apoptosis compared with negative control groups by flow cytometry detection. (B) Quantification of flow cytometry data. Quadrant R2 and X4 were apoptotic cells. Data are presented as the mean ± standard deviation for three independent experiments. **P<0.01. RNF138, ring finger protein 138; siRNA, small interfering RNA; 7-AAD, 7-aminoactinomycin D.

Article Snippet: Bcl2 associated X apoptosis regulator (BAX; 1:1,000; cat. no. sc-4239 WB; Santa Cruz Biotechnology, Inc.), apoptosis regulator Bcl2 (Bcl2; 1:1,000; cat. no. sc-23960; Santa Cruz Biotechnology, Inc.); vascular endothelial growth factor (VEGF; 1:1,000; cat. no. sc-365578) and hypoxia-inducible factor-1α (HIF-1α; 1:1,000; cat. no. sc-13515); Santa Cruz Biotechnology, Inc.), cleaved caspase3 (cat. no. 9664), caspase3 (cat. no. 9662), Erk1/2 (cat. no. 4695), phospho (p)-Erk1/2 (cat. no. 4370), matrix metalloproteinase 2 (MMP2; cat. no. 40994), E-cadherin (cat. no. 14472) and vimentin (cat. no. 5741) were acquired from Cell Signaling Technology, Inc. and RNF138 (cat. no. DF4025; Affinity Biosciences, Cincinnati, OH, USA) at a dilution of 1:1,500.

Techniques: Transduction, Negative Control, Transfection, Flow Cytometry, Standard Deviation, Small Interfering RNA

Journal: Oncology Reports

Article Title: Downregulation of RNF138 inhibits cellular proliferation, migration, invasion and EMT in glioma cells via suppression of the Erk signaling pathway

doi: 10.3892/or.2018.6744

Figure Lengend Snippet: Protein levels of apoptotic-associated factors in U87 and U251 cells transduced with RNF138-siRNA and negative control lentivirus. (A) Expression of caspase3, cleaved caspase3, BAX and Bcl2 were detected by western blot analysis. β-actin served as the loading control. Densitometry analysis of (B) caspase3, (C) cleaved caspase3, (D) BAX and (E) Bcl2, relative to β-actin expression in U87 and U251 cells with ImageJ analysis. Data are presented as the mean ± standard deviation for three independent experiments. **P<0.01. RNF138, ring finger protein 138; siRNA, small interfering RNA; BAX, Bcl2 associated X apoptosis regulator; Bcl2, Bcl2 apoptosis regulator; NS, not significant.

Article Snippet: Bcl2 associated X apoptosis regulator (BAX; 1:1,000; cat. no. sc-4239 WB; Santa Cruz Biotechnology, Inc.), apoptosis regulator Bcl2 (Bcl2; 1:1,000; cat. no. sc-23960; Santa Cruz Biotechnology, Inc.); vascular endothelial growth factor (VEGF; 1:1,000; cat. no. sc-365578) and hypoxia-inducible factor-1α (HIF-1α; 1:1,000; cat. no. sc-13515); Santa Cruz Biotechnology, Inc.), cleaved caspase3 (cat. no. 9664), caspase3 (cat. no. 9662), Erk1/2 (cat. no. 4695), phospho (p)-Erk1/2 (cat. no. 4370), matrix metalloproteinase 2 (MMP2; cat. no. 40994), E-cadherin (cat. no. 14472) and vimentin (cat. no. 5741) were acquired from Cell Signaling Technology, Inc. and RNF138 (cat. no. DF4025; Affinity Biosciences, Cincinnati, OH, USA) at a dilution of 1:1,500.

Techniques: Transduction, Negative Control, Expressing, Western Blot, Control, Standard Deviation, Small Interfering RNA