mib2 1  (Addgene inc)

Journal: Oncology Letters

Article Title: Ectopic expression of E3 ubiquitin-protein ligase 2 in glioma and enhances resistance to apoptosis through activating nuclear factor κ-light-chain-enhancer of B cells

doi: 10.3892/ol.2018.9153

Figure Lengend Snippet: Clinicopathological characteristics of patient samples and expression of MIB2 in glioma.

Article Snippet: The pNF-κB-Luc plasmids were purchased from the Clontech Laboratories, Inc. (Mountainview, CA, USA), and the Flag-MIB2 plasmids used for MIB2 overexpression were purchased from Addgene, Inc. (Cambridge, MA, USA).

Techniques: Expressing

Journal: Oncology Letters

Article Title: Ectopic expression of E3 ubiquitin-protein ligase 2 in glioma and enhances resistance to apoptosis through activating nuclear factor κ-light-chain-enhancer of B cells

doi: 10.3892/ol.2018.9153

Figure Lengend Snippet: Expression of MIB2 is elevated in human glioma cell lines and clinical glioma specimens. (A) RT-qPCR analysis of MIB2 mRNA in NHA and glioma T98G, LN-18 and A172 cell lines. Data are normalized to GAPDH and are presented as the mean ± standard deviation of 3 independent experiments. **P<0.01 by one-way ANOVA and Tukey's post hoc test. (B) Expression of MIB2 protein in NHA and indicated glioma cell lines. α-Tubulin was used as the loading control. (C) Reverse transcription-quantitative polymerase chain reaction analysis of MIB2 mRNA in four pairs of primary glioma tissues. Data are normalized to GAPDH and are presented as the mean ± SEM of three experiments, with statistical significance determined by one-way ANOVA and Tukey's post hoc test. **P<0.01. (D) Expression of MIB2 protein in paired T and N samples by western blot analysis. α-Tubulin was used as the loading control. NHA, normal human astrocyte; MIB2, E3 ubiquitin-protein ligase; ANOVA, analysis of variance; T, primary glioma samples; N, adjacent non-cancerous brain tissues.

Article Snippet: The pNF-κB-Luc plasmids were purchased from the Clontech Laboratories, Inc. (Mountainview, CA, USA), and the Flag-MIB2 plasmids used for MIB2 overexpression were purchased from Addgene, Inc. (Cambridge, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Ubiquitin Proteomics

Journal: Oncology Letters

Article Title: Ectopic expression of E3 ubiquitin-protein ligase 2 in glioma and enhances resistance to apoptosis through activating nuclear factor κ-light-chain-enhancer of B cells

doi: 10.3892/ol.2018.9153

Figure Lengend Snippet: Immunohistochemical analysis of MIB2 expression in normal brain tissues and primary glioma tumors. (A) Representative images from IHC assays of normal brain tissues and specimens of 69 archived glioma cases. (a,b) Normal brain tissue; (c,d) WHO grade 1 pilocytic astrocytoma; (e,f) WHO grade 2 diffuse astrocytoma; (g,h) WHO grade 3 anaplastic astrocytoma; (i,j) WHO grade 4 glioblastoma multiforme. (a,c,e,g,i) Magnification, ×200. (b,d,f,h and j) Magnification, ×400. (B) Comparative statistical quantification of the mean values of MIB2 staining between normal brain tissues (n=3) and glioma specimens of different WHO grades. Student's t-test was used for statistical analysis. **P<0.01. IHC, immunohistochemistry; MIB2, E3 ubiquitin-protein ligase; WHO, World Health Organization.

Article Snippet: The pNF-κB-Luc plasmids were purchased from the Clontech Laboratories, Inc. (Mountainview, CA, USA), and the Flag-MIB2 plasmids used for MIB2 overexpression were purchased from Addgene, Inc. (Cambridge, MA, USA).

Techniques: Immunohistochemical staining, Expressing, Staining, Immunohistochemistry, Ubiquitin Proteomics

Journal: Oncology Letters

Article Title: Ectopic expression of E3 ubiquitin-protein ligase 2 in glioma and enhances resistance to apoptosis through activating nuclear factor κ-light-chain-enhancer of B cells

doi: 10.3892/ol.2018.9153

Figure Lengend Snippet: Overexpression of MIB2 enhances the anti-apoptotic activity of glioma cells. (A) Overexpression of MIB2 in the glioma T98G cell line. α-Tubulin was used as the loading control. (B) Overexpression of MIB2 inhibits cell death induced by UV irradiation. MIB2-overexpressing glioma cell lines were treated by UV irradiation (20 and 40 J/m 2 ), and viable cells were counted using the trypan blue exclusion assay. Data are presented as the mean ± standard error of the mean of three experiments with statistical significance determined by one-way analysis of variance with Tukey's post hoc test. **P<0.01. (C) MIB2 reduces UV-induced apoptosis of glioma cells. MIB2-overexpressing glioma cell lines were treated with UV irradiation (40 J/m 2 ), followed by Annexin V-fluorescein isothiocyanate and PI staining, and the number of Annexin V+/PI-cells was counted from 5 random fields. Results are expressed as percentages of total cells. Student's t-test was used for statistical analysis. **P<0.01. (D) MIB2-overexpressing glioma cell lines were treated with UV irradiation (40 J/m 2 ) and harvested for cell lysate preparation. Western blotting was performed to assess the levels of cleaved caspase-3 and Bcl-2 protein. α-Tubulin was used as the loading control. UV, ultraviolet; MIB2, E3 ubiquitin-protein ligase; PI, propidium iodide; Bcl-2, B-cell lymphoma 2.

Article Snippet: The pNF-κB-Luc plasmids were purchased from the Clontech Laboratories, Inc. (Mountainview, CA, USA), and the Flag-MIB2 plasmids used for MIB2 overexpression were purchased from Addgene, Inc. (Cambridge, MA, USA).

Techniques: Over Expression, Activity Assay, Control, Irradiation, Trypan Blue Exclusion Assay, Staining, Western Blot, Ubiquitin Proteomics

Journal: Oncology Letters

Article Title: Ectopic expression of E3 ubiquitin-protein ligase 2 in glioma and enhances resistance to apoptosis through activating nuclear factor κ-light-chain-enhancer of B cells

doi: 10.3892/ol.2018.9153

Figure Lengend Snippet: Knockdown of endogenous MIB2 attenuates the resistance of glioma cells to apoptosis. (A) Knockdown of MIB2 in glioma cell lines was analyzed by WB. α-tubulin was used as the loading control. (B) Knockdown of MIB2 increases cell death. The indicated cells were treated by UV irradiation (20 and 40 J/m 2 ) and counted for viable cells using the trypan blue exclusion assay. Data are presented as the mean ± standard error of the mean of three experiments with statistical significance determined by one-way analysis of variance with Tukey's post hoc test. **P<0.01. (C) Knockdown of MIB2 enhances the sensitivity of glioma cells to UV-induced apoptosis. MIB2-knockdown glioma cell lines were treated with UV irradiation (40 J/m 2 ), followed by Annexin V-fluorescein isothiocyanate and PI staining, and the number of Annexin V+/PI-cells was counted from 5 random fields. Results are expressed as percentages of total cells. Columns represent the mean of three experiments. A Student's t-test was used for statistical analysis. **P<0.01. (D) T98G cells were treated with UV irradiation (40 J/m 2 ) and were harvested for cell lysate preparation. WB was performed to assess the levels of cleaved caspase-3 and Bcl-2 protein. α-Tubulin was used as the loading control. WB, western blotting; MIB2, E3 ubiquitin-protein ligase; PI, propidium iodide; UV, ultraviolet; Bcl-2, B-cell lymphoma 2.

Article Snippet: The pNF-κB-Luc plasmids were purchased from the Clontech Laboratories, Inc. (Mountainview, CA, USA), and the Flag-MIB2 plasmids used for MIB2 overexpression were purchased from Addgene, Inc. (Cambridge, MA, USA).

Techniques: Knockdown, Control, Irradiation, Trypan Blue Exclusion Assay, Staining, Western Blot, Ubiquitin Proteomics

Journal: Oncology Letters

Article Title: Ectopic expression of E3 ubiquitin-protein ligase 2 in glioma and enhances resistance to apoptosis through activating nuclear factor κ-light-chain-enhancer of B cells

doi: 10.3892/ol.2018.9153

Figure Lengend Snippet: MIB2 activates NF-κB signaling in glioma cells. (A) Activation of NF-κB-dependent reporter gene by overexpression of MIB2. Cells co-transfected with 1 µg pNF-κB-luciferase and 10 ng pRL-TK Renilla plasmids for 24 h in 12-well plates were lysed and the luciferase activity was measured. Data are presented as the mean of three independent experiments. Student's t-test was used for statistical analysis. **P<0.01. (B) Downregulation of NF-κB-dependent reporter gene by knockdown of MIB2. Cells co-transfected with 1 µg pNF-κB-luciferase and 10 ng pRL-TK Renilla plasmids for 24 h in 12-well plates were lysed and the luciferase activity was measured. Data are presented as the mean of three independent experiments. Student's t-test was used for statistical analysis. **P<0.01. (C) Western blot analysis of NF-κB p65 phosphorylation in MIB2-overexpressing glioma cell lines. α-Tubulin was used as the loading control. (D) Western blot analysis of NF-κB p65 phosphorylation in MIB2-knockdown glioma cell lines. α-Tubulin was used as the loading control. MIB2, E3 ubiquitin-protein ligase; p, phosphorylated; NF-κB, nuclear factor κ-light-chain-enhancer of B cells; p65, NF-κB p65 subunit.

Article Snippet: The pNF-κB-Luc plasmids were purchased from the Clontech Laboratories, Inc. (Mountainview, CA, USA), and the Flag-MIB2 plasmids used for MIB2 overexpression were purchased from Addgene, Inc. (Cambridge, MA, USA).

Techniques: Activation Assay, Over Expression, Transfection, Luciferase, Activity Assay, Knockdown, Western Blot, Phospho-proteomics, Control, Ubiquitin Proteomics