Review

ng2 sirna (Santa Cruz Biotechnology)

Name: ng2 sirna
Brand: Santa Cruz Biotechnology
Rating: 93 (2 reviews)

Santa Cruz Biotechnology is a verified supplier

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

Santa Cruz Biotechnology ng2 sirna
Ng2 Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/ng2 sirna/product/Santa Cruz Biotechnology
Average 93 stars, based on 2 article reviews

ng2 sirna - by Bioz Stars, 2026-02

93/100 stars

Images

Image Search Results

CK2 inhibition reduces NG2 expression in human GBM cell lines. ( A ) A1207 and U87 cells were treated with vehicle (DMSO) or CX-4945 (10 µM) for 72 h. The cells were lysed and the expression of NG2, Akt, pAkt S129 , CK2α, CK2α’, CK2β and α-tubulin (as loading control) was analyzed by western blot. ( B – E ) A1207 and U87 cells were treated as described in ( A ) and the expression of pAkt/Akt ( B , C ) and NG2 ( D , E ) was quantitatively analyzed. Vehicle-treated cells were set 100%. Mean ± SD. * p < 0.05 vs. vehicle ( n = 3). ( F , G ) A1207 and U87 cells were treated as described in ( A ), scratched and the mean fluorescence intensity (MFI) of NG2-positive cells was assessed by flow cytometry. The MFI of vehicle-treated cells was set 100%. Mean ± SD. * p < 0.05 vs. vehicle ( n = 4). ( H ) A1207 and U87 wild type and CK2α KO cells were lysed and the expression of NG2, Akt, pAkt S129 , CK2α, CK2α’, CK2β and α-tubulin (as loading control) was analyzed by western blot. ( I – N ) A1207 and U87 cells were treated as described in ( H ) and the expression of CK2α ( I , J ), pAkt/Akt ( K , L ) and NG2 ( M , N ) was quantitatively assessed. Wild type cells were set 100%. Mean ± SD. * p < 0.05 vs. wild type (A1207: n = 4; U87: n = 5). ( O ) A1207 wild type and CK2α KO cells were lysed and the expression of FAK, pFAK and α-tubulin (as loading control) was analyzed by western blot. ( P ) A1207 wild type and CK2α KO cells were lysed and the expression of pFAK/FAK was quantitatively assessed. Wild type cells were set 100%. Mean ± SD. * p < 0.05 vs. wild type ( n = 3).

Journal: Cancers

Article Title: CK2 Activity Mediates the Aggressive Molecular Signature of Glioblastoma Multiforme by Inducing Nerve/Glial Antigen (NG)2 Expression

doi: 10.3390/cancers13071678

Figure Lengend Snippet: CK2 inhibition reduces NG2 expression in human GBM cell lines. ( A ) A1207 and U87 cells were treated with vehicle (DMSO) or CX-4945 (10 µM) for 72 h. The cells were lysed and the expression of NG2, Akt, pAkt S129 , CK2α, CK2α’, CK2β and α-tubulin (as loading control) was analyzed by western blot. ( B – E ) A1207 and U87 cells were treated as described in ( A ) and the expression of pAkt/Akt ( B , C ) and NG2 ( D , E ) was quantitatively analyzed. Vehicle-treated cells were set 100%. Mean ± SD. * p < 0.05 vs. vehicle ( n = 3). ( F , G ) A1207 and U87 cells were treated as described in ( A ), scratched and the mean fluorescence intensity (MFI) of NG2-positive cells was assessed by flow cytometry. The MFI of vehicle-treated cells was set 100%. Mean ± SD. * p < 0.05 vs. vehicle ( n = 4). ( H ) A1207 and U87 wild type and CK2α KO cells were lysed and the expression of NG2, Akt, pAkt S129 , CK2α, CK2α’, CK2β and α-tubulin (as loading control) was analyzed by western blot. ( I – N ) A1207 and U87 cells were treated as described in ( H ) and the expression of CK2α ( I , J ), pAkt/Akt ( K , L ) and NG2 ( M , N ) was quantitatively assessed. Wild type cells were set 100%. Mean ± SD. * p < 0.05 vs. wild type (A1207: n = 4; U87: n = 5). ( O ) A1207 wild type and CK2α KO cells were lysed and the expression of FAK, pFAK and α-tubulin (as loading control) was analyzed by western blot. ( P ) A1207 wild type and CK2α KO cells were lysed and the expression of pFAK/FAK was quantitatively assessed. Wild type cells were set 100%. Mean ± SD. * p < 0.05 vs. wild type ( n = 3).

Article Snippet: Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbecco’s Modified Eagle’s Medium (DMEM), Lipofectamine3000 reagent, Opti-MEM Reduced Serum Medium (Gibco), fetal calf serum (FCS), penicillin-streptomycin and small interfering RNA (siRNA) duplexes directed against NG2 (ID: 146.147) were from Thermo Fisher Scientific (Karlsruhe, Germany).

Techniques: Inhibition, Expressing, Control, Western Blot, Fluorescence, Flow Cytometry

CK2 inhibition reduces NG2 gene expression in human GBM cell lines. ( A ) A1207 wild type and CK2α KO cells were transfected with pGL4-NG2 p1.2.4.1 , cultivated for 24 h, lysed and the transcriptional activity was detected by a luciferase assay. Relative luciferase units (RLU) of wild type cells were used as control. Mean ± SD. * p < 0.05 vs. wild type ( n = 4). ( B ) A1207 cells were transfected with pGL4-NG2 p1.2.4.1 for 24 h, treated with vehicle (DMSO) or CX-4945 (10 µM) and analyzed by a luciferase assay. RLU of vehicle-treated cells were used as control. Mean ± SD. * p < 0.05 vs. vehicle ( n = 3). ( C , D ) A1207 and U87 cells were treated with vehicle (DMSO) or CX-4945 (10 µM) for 72 h, harvested and total RNA was isolated. The relative gene expression of NG2 was examined by qRT-PCR normalized to GAPDH. Vehicle-treated cells were set 100%. Mean ± SD. * p < 0.05 vs. vehicle ( n = 4). ( E , F ) A1207 and U87 cells were treated with vehicle (DMSO) or CX-4945 (10 µM) in the presence of cycloheximide. The cells were scratched after 0 h, 24 h, 48 h and 72 h and the MFI of NG2-positive cells was assessed by flow cytometry. MFI of cells at 0 h was set 100%. Mean ± SD ( n = 4).

Journal: Cancers

Article Title: CK2 Activity Mediates the Aggressive Molecular Signature of Glioblastoma Multiforme by Inducing Nerve/Glial Antigen (NG)2 Expression

doi: 10.3390/cancers13071678

Figure Lengend Snippet: CK2 inhibition reduces NG2 gene expression in human GBM cell lines. ( A ) A1207 wild type and CK2α KO cells were transfected with pGL4-NG2 p1.2.4.1 , cultivated for 24 h, lysed and the transcriptional activity was detected by a luciferase assay. Relative luciferase units (RLU) of wild type cells were used as control. Mean ± SD. * p < 0.05 vs. wild type ( n = 4). ( B ) A1207 cells were transfected with pGL4-NG2 p1.2.4.1 for 24 h, treated with vehicle (DMSO) or CX-4945 (10 µM) and analyzed by a luciferase assay. RLU of vehicle-treated cells were used as control. Mean ± SD. * p < 0.05 vs. vehicle ( n = 3). ( C , D ) A1207 and U87 cells were treated with vehicle (DMSO) or CX-4945 (10 µM) for 72 h, harvested and total RNA was isolated. The relative gene expression of NG2 was examined by qRT-PCR normalized to GAPDH. Vehicle-treated cells were set 100%. Mean ± SD. * p < 0.05 vs. vehicle ( n = 4). ( E , F ) A1207 and U87 cells were treated with vehicle (DMSO) or CX-4945 (10 µM) in the presence of cycloheximide. The cells were scratched after 0 h, 24 h, 48 h and 72 h and the MFI of NG2-positive cells was assessed by flow cytometry. MFI of cells at 0 h was set 100%. Mean ± SD ( n = 4).

Techniques: Inhibition, Gene Expression, Transfection, Activity Assay, Luciferase, Control, Isolation, Quantitative RT-PCR, Flow Cytometry

CK2 inhibition reduces the migratory capacity of NG2-positive GBM cell lines. ( A ) A1207 cells were transfected with ctrl si or NG2 si for 72 h. The cells were harvested, lysed and the expression of NG2, CK2α and α-tubulin (as loading control) was analyzed by western blot. ( B ) A1207 cells were treated as described in ( A ), detached and their migration was assessed by transwell assays (scale bar: 50 µm). ( C ) A1207 cells were treated as described in ( A ) and the migration was quantitatively assessed. Ctrl si-transfected cells were used as control. Mean ± SD. * p < 0.05 vs. ctrl si ( n = 4). ( D ) A1207 cells were treated with vehicle (DMSO) or CX-4945 (10 µM) for 72 h, detached and their migration was assessed by transwell assays (scale bar: 50 µm). ( E ) A1207 cells were treated as described in ( D ) and migration was quantitatively assessed. Vehicle-treated cells were used as control. Mean ± SD. * p < 0.05 vs. vehicle ( n = 4). ( F ) A1207 were transfected with mock (pEF6 vector) or NG2 plasmid and incubated for 48 h. Expression of NG2, CK2α and α-tubulin (as loading control) was analyzed by western blot. ( G ) A1207 cells were transfected as described in ( F ), treated with CX-4945 for 24 h, detached and their migration was assessed by transwell assays (scale bar: 50 µm). ( H ) A1207 cells were transfected and treated as described in ( G ) and migration was quantitatively assessed. Mock-transfected cells were used as control. Mean ± SD. * p < 0.05 vs. mock ( n = 4).

Journal: Cancers

Article Title: CK2 Activity Mediates the Aggressive Molecular Signature of Glioblastoma Multiforme by Inducing Nerve/Glial Antigen (NG)2 Expression

doi: 10.3390/cancers13071678

Figure Lengend Snippet: CK2 inhibition reduces the migratory capacity of NG2-positive GBM cell lines. ( A ) A1207 cells were transfected with ctrl si or NG2 si for 72 h. The cells were harvested, lysed and the expression of NG2, CK2α and α-tubulin (as loading control) was analyzed by western blot. ( B ) A1207 cells were treated as described in ( A ), detached and their migration was assessed by transwell assays (scale bar: 50 µm). ( C ) A1207 cells were treated as described in ( A ) and the migration was quantitatively assessed. Ctrl si-transfected cells were used as control. Mean ± SD. * p < 0.05 vs. ctrl si ( n = 4). ( D ) A1207 cells were treated with vehicle (DMSO) or CX-4945 (10 µM) for 72 h, detached and their migration was assessed by transwell assays (scale bar: 50 µm). ( E ) A1207 cells were treated as described in ( D ) and migration was quantitatively assessed. Vehicle-treated cells were used as control. Mean ± SD. * p < 0.05 vs. vehicle ( n = 4). ( F ) A1207 were transfected with mock (pEF6 vector) or NG2 plasmid and incubated for 48 h. Expression of NG2, CK2α and α-tubulin (as loading control) was analyzed by western blot. ( G ) A1207 cells were transfected as described in ( F ), treated with CX-4945 for 24 h, detached and their migration was assessed by transwell assays (scale bar: 50 µm). ( H ) A1207 cells were transfected and treated as described in ( G ) and migration was quantitatively assessed. Mock-transfected cells were used as control. Mean ± SD. * p < 0.05 vs. mock ( n = 4).

Techniques: Inhibition, Transfection, Expressing, Control, Western Blot, Migration, Plasmid Preparation, Incubation

CK2 and NG2 mRNA expression positively correlate in human GBM. ( A – D ) The relative mRNA expression (TCGA-based data; arbitrary units (AU)) of NG2 ( A ), CK2α ( B ), CK2α’ ( C ) and CK2β ( D ) was analyzed in human astrocytoma (grade I-II), oligodendroglioma (grade II), anaplastic oligoastrocytoma and astrocytoma (grade III) as well as in GBM (grade IV). * p < 0.05 vs. GBM ( n = 682). ( E – G ) Spearman correlations of NG2 mRNA expression (AU) with CK2α ( E ), CK2α’ ( F ) and CK2β ( G ) mRNA expression (TCGA-based data, AU, n = 141).

Journal: Cancers

Article Title: CK2 Activity Mediates the Aggressive Molecular Signature of Glioblastoma Multiforme by Inducing Nerve/Glial Antigen (NG)2 Expression

doi: 10.3390/cancers13071678

Figure Lengend Snippet: CK2 and NG2 mRNA expression positively correlate in human GBM. ( A – D ) The relative mRNA expression (TCGA-based data; arbitrary units (AU)) of NG2 ( A ), CK2α ( B ), CK2α’ ( C ) and CK2β ( D ) was analyzed in human astrocytoma (grade I-II), oligodendroglioma (grade II), anaplastic oligoastrocytoma and astrocytoma (grade III) as well as in GBM (grade IV). * p < 0.05 vs. GBM ( n = 682). ( E – G ) Spearman correlations of NG2 mRNA expression (AU) with CK2α ( E ), CK2α’ ( F ) and CK2β ( G ) mRNA expression (TCGA-based data, AU, n = 141).

Techniques: Expressing

CK2 inhibition reduces NG2 expression in patient-derived GBM cells. ( A ) Patient-derived GBM cells (T8399, T8478, T8475 and T8470) were treated with vehicle (DMSO) or CX-4945 (10 µM) for 72 h. Subsequently, the cells were lysed and the expression of NG2, Akt, pAkt S129 , CK2α, CK2α’ and β-actin (as loading control) was analyzed by western blot. ( B , C ) The cells were treated as described in (A) and the expression of pAkt/Akt ( B ) and NG2 ( C ) was quantitatively analyzed. Vehicle-treated cells were set 100%. ( D ) Patient-derived GBM cells (T8399, T8478, T8475 and T8470) were treated with vehicle (DMSO) or CX-4945 (10 µM) for 72 h, scratched and the MFI of NG2-positive cells was assessed by flow cytometry. Vehicle-treated cells were set 100%.

Journal: Cancers

Article Title: CK2 Activity Mediates the Aggressive Molecular Signature of Glioblastoma Multiforme by Inducing Nerve/Glial Antigen (NG)2 Expression

doi: 10.3390/cancers13071678

Figure Lengend Snippet: CK2 inhibition reduces NG2 expression in patient-derived GBM cells. ( A ) Patient-derived GBM cells (T8399, T8478, T8475 and T8470) were treated with vehicle (DMSO) or CX-4945 (10 µM) for 72 h. Subsequently, the cells were lysed and the expression of NG2, Akt, pAkt S129 , CK2α, CK2α’ and β-actin (as loading control) was analyzed by western blot. ( B , C ) The cells were treated as described in (A) and the expression of pAkt/Akt ( B ) and NG2 ( C ) was quantitatively analyzed. Vehicle-treated cells were set 100%. ( D ) Patient-derived GBM cells (T8399, T8478, T8475 and T8470) were treated with vehicle (DMSO) or CX-4945 (10 µM) for 72 h, scratched and the MFI of NG2-positive cells was assessed by flow cytometry. Vehicle-treated cells were set 100%.

Techniques: Inhibition, Expressing, Derivative Assay, Control, Western Blot, Flow Cytometry

$A ) Yeast two hybrid screen: Yeast cells were transformed with the serine protease OMI/HtrA2 and positive clones defined by ß-Gal-expression. Selection was on Trp-, Leu- and His-deficient media. The NG2 PDZ-binding motif and different mutants (red letters) of this domain were tested for binding. B) Lysates of Oli-neu cells and HEK-293T cells (as a control) were immunoprecipitated with pcOMI/HtrA2 antibody and immunoblotted as indicated. Endogenous NG2 is coimmunoprecipitated with the OMI/HtrA2-IP. As a negative control, the mitochondrial membrane protein COX-1 was analysed. C) Lysates of Oli-neu cells and HEK-293T cells were immunoprecipitated with monoclonal NG2 antibody and immunoblotted as indicated. Endogenous OMI/Htra2, mainly the processed 37 kDa form, is coimmunoprecipitated with the immunoprecipitated NG2-IP. As a negative control, the mitochondrial membrane protein COX-1 was analysed. In the pre-clear fraction, beads without the antibodies served as additional controls.$

Journal: PLoS ONE

Article Title: The NG2 Proteoglycan Protects Oligodendrocyte Precursor Cells against Oxidative Stress via Interaction with OMI/HtrA2

doi: 10.1371/journal.pone.0137311

Figure Lengend Snippet: A ) Yeast two hybrid screen: Yeast cells were transformed with the serine protease OMI/HtrA2 and positive clones defined by ß-Gal-expression. Selection was on Trp-, Leu- and His-deficient media. The NG2 PDZ-binding motif and different mutants (red letters) of this domain were tested for binding. B) Lysates of Oli-neu cells and HEK-293T cells (as a control) were immunoprecipitated with pcOMI/HtrA2 antibody and immunoblotted as indicated. Endogenous NG2 is coimmunoprecipitated with the OMI/HtrA2-IP. As a negative control, the mitochondrial membrane protein COX-1 was analysed. C) Lysates of Oli-neu cells and HEK-293T cells were immunoprecipitated with monoclonal NG2 antibody and immunoblotted as indicated. Endogenous OMI/Htra2, mainly the processed 37 kDa form, is coimmunoprecipitated with the immunoprecipitated NG2-IP. As a negative control, the mitochondrial membrane protein COX-1 was analysed. In the pre-clear fraction, beads without the antibodies served as additional controls.

Article Snippet: The siRNA against NG2 (target sequence in the UTR1) and the control-siRNA (target sequence: 5′-AAT TCT CCG AAC GTG TCA CGT-3′ ) were obtained from QIAGEN.

Techniques: Two Hybrid Screening, Transformation Assay, Clone Assay, Expressing, Selection, Binding Assay, Control, Immunoprecipitation, Negative Control, Membrane

A) Mito-Capture dye fluoresces green in the cytosol. In intact mitochondria with normal membrane potentials the dye fluoresces red. With increasing concentrations of H 2 O 2 for four hours resulting in oxidative stress, the mitochondrial membrane potential is disrupted and the red fluorescence is diminished. B) Scheme of the CoIP ELISA: living cells were bound to plate with the monoclonal NG2 antibody and then cultured for five hours in the presence of H 2 O 2 . The cells were then lysed and OMI/HtrA2 that was released from mitochondria into the cytosol and bound to the NG2 proteoglycan was detected with polyclonal OMI/HtrA2 antibody and horseradish peroxidase-coupled anti-rabbit antibody. C) Binding of OMI/HtrA2 to NG2 is detected at different concentrations of hydrogen peroxide and compared to unstressed control cells, which are set as 0%. Pooled data from three experiments are shown (n = 3, SEM, unpaired Student’s t-test: p-values ***<0.001; **<0.01; *<0.05). In controls lacking plate-bound NG2 antibody, increased levels of oxidative stress do not significantly alter the background signal detected with the OMI and HRP-coupled antibodies.

Journal: PLoS ONE

Article Title: The NG2 Proteoglycan Protects Oligodendrocyte Precursor Cells against Oxidative Stress via Interaction with OMI/HtrA2

doi: 10.1371/journal.pone.0137311

Figure Lengend Snippet: A) Mito-Capture dye fluoresces green in the cytosol. In intact mitochondria with normal membrane potentials the dye fluoresces red. With increasing concentrations of H 2 O 2 for four hours resulting in oxidative stress, the mitochondrial membrane potential is disrupted and the red fluorescence is diminished. B) Scheme of the CoIP ELISA: living cells were bound to plate with the monoclonal NG2 antibody and then cultured for five hours in the presence of H 2 O 2 . The cells were then lysed and OMI/HtrA2 that was released from mitochondria into the cytosol and bound to the NG2 proteoglycan was detected with polyclonal OMI/HtrA2 antibody and horseradish peroxidase-coupled anti-rabbit antibody. C) Binding of OMI/HtrA2 to NG2 is detected at different concentrations of hydrogen peroxide and compared to unstressed control cells, which are set as 0%. Pooled data from three experiments are shown (n = 3, SEM, unpaired Student’s t-test: p-values ***<0.001; **<0.01; *<0.05). In controls lacking plate-bound NG2 antibody, increased levels of oxidative stress do not significantly alter the background signal detected with the OMI and HRP-coupled antibodies.

Article Snippet: The siRNA against NG2 (target sequence in the UTR1) and the control-siRNA (target sequence: 5′-AAT TCT CCG AAC GTG TCA CGT-3′ ) were obtained from QIAGEN.

Techniques: Membrane, Fluorescence, Enzyme-linked Immunosorbent Assay, Cell Culture, Binding Assay, Control

A) MTT-assay and LDH-assay with Oli-neu cells transfected with NG2siRNA and control siRNA and stressed with H 2 O 2 . MTT conversion is displayed in comparison to unstressed controls, which are set as 100% (n = 7, SEM, unpaired Student’s t-test: p-values **<0.01; *<0.05) are shown. The knockdown efficiency is shown by Western Blots for NG2 (insert). Cells are stressed for 4h. LDH release is displayed in comparison to unstressed controls, which are set as 100% (n = 4, SEM, unpaired Student’s t-test: p-values *<0.05) B) MTT-assay and LDH-assay with Oli-neu cells transfected with NG2siRNA and control siRNA and stressed with ONOO - (peroxynitrite). MTT conversion and LDH release is displayed in comparison to unstressed controls, which are set as 100% (n = 3, SEM, unpaired Student’s t-test: p-values *<0.05) are shown. C) Western Blot analysis of PARP levels normalised against Tubulin in cells with and without NG2 knockdown relative to unstressed controls which are set as 100% (n = 3, SEM, unpaired Student’s t-Test: p-value **<0.01). Western Blots from one single experiment are shown as an example. D) NG2siRNA and control siRNA transfected Oli-neu cells stressed with 50μM H 2 O 2 in comparison to unstressed controls. Triple staining with DAPI ( blue ), cleaved-caspase-3 ( red ) and monoclonal antibody recognising NG2 ( green ): the enlarged inserts show a cleaved-caspase-3-positive cell with a pyknotic nucleus (DAPI staining). Scale bar = 50μm.

Journal: PLoS ONE

Article Title: The NG2 Proteoglycan Protects Oligodendrocyte Precursor Cells against Oxidative Stress via Interaction with OMI/HtrA2

doi: 10.1371/journal.pone.0137311

Figure Lengend Snippet: A) MTT-assay and LDH-assay with Oli-neu cells transfected with NG2siRNA and control siRNA and stressed with H 2 O 2 . MTT conversion is displayed in comparison to unstressed controls, which are set as 100% (n = 7, SEM, unpaired Student’s t-test: p-values **<0.01; *<0.05) are shown. The knockdown efficiency is shown by Western Blots for NG2 (insert). Cells are stressed for 4h. LDH release is displayed in comparison to unstressed controls, which are set as 100% (n = 4, SEM, unpaired Student’s t-test: p-values *<0.05) B) MTT-assay and LDH-assay with Oli-neu cells transfected with NG2siRNA and control siRNA and stressed with ONOO - (peroxynitrite). MTT conversion and LDH release is displayed in comparison to unstressed controls, which are set as 100% (n = 3, SEM, unpaired Student’s t-test: p-values *<0.05) are shown. C) Western Blot analysis of PARP levels normalised against Tubulin in cells with and without NG2 knockdown relative to unstressed controls which are set as 100% (n = 3, SEM, unpaired Student’s t-Test: p-value **<0.01). Western Blots from one single experiment are shown as an example. D) NG2siRNA and control siRNA transfected Oli-neu cells stressed with 50μM H 2 O 2 in comparison to unstressed controls. Triple staining with DAPI ( blue ), cleaved-caspase-3 ( red ) and monoclonal antibody recognising NG2 ( green ): the enlarged inserts show a cleaved-caspase-3-positive cell with a pyknotic nucleus (DAPI staining). Scale bar = 50μm.

Article Snippet: The siRNA against NG2 (target sequence in the UTR1) and the control-siRNA (target sequence: 5′-AAT TCT CCG AAC GTG TCA CGT-3′ ) were obtained from QIAGEN.

Techniques: MTT Assay, Lactate Dehydrogenase Assay, Transfection, Control, Comparison, Knockdown, Western Blot, Staining

A) Quantification of Western Blot analysis of lysates of cerebellum cultures containing all cell types of WT and NG2-KO mice with the specific OMI-inhibitor UCF101 (grey columns) or without (black columns). Cultures were exposed to H 2 O 2 for 18 hours. OPC were quantified by blotting for NG2 (WT), EYFP (KO) or JAM-A (for WT and KO). Degradation of PARP is an indication of cell death. In the presence of the OMI inhibitor UCF101, less death of NG2-lacking (NG2-KO) OPC is observed. The WB signals of samples from six individual animals (3WT; 3NG2-KO) with the indicated antibodies are shown on the right from which the quantitative data (bar graphs) is derived. All signals are normalised to Tubulin and compared to the unstressed controls (set as 100%) (n = 3, SEM, unpaired Student’s t-Test: p-value **<0.01; *<0.05). B) Cerebellum cultures of wildtype (WT) mice and NG2/EYFP knockout mice (NG2-KO) were incubated with 800μm H 2 O 2 for 18 hours, lysed and compared to unstressed controls by WB with cell-type-specific markers. Blots are shown of one experiment with three replicates. C) Quantification of WB signals for different cell-type specific markers against Tubulin of the cerebellum cultures, compared to unstressed cultures (EYFP and NG2 respectively for OPC, ASPA for oligodendrocytes, δ-Enolase for neurons and GFAP for astrocytes; (n = 4, SEM, unpaired Student’s t-Test: p-value *<0.05) as shown in the example (B). D) WB analyses with quantification of activated caspase-3 (cleaved caspase-3) of WT and NG2-KO cerebellum cultures with and without oxidative stress for 5 hours (n = 1). An example of apoptotic cell death in OPC in the cerebellum cultures from the NG2-KO mice after 5 hours of stress with 800μm H 2 O 2 stained with cleaved-caspase-3 (red), EYFP (green) and DAPI (blue) is shown. Scale bar = 50μm.

Journal: PLoS ONE

Article Title: The NG2 Proteoglycan Protects Oligodendrocyte Precursor Cells against Oxidative Stress via Interaction with OMI/HtrA2

doi: 10.1371/journal.pone.0137311

Figure Lengend Snippet: A) Quantification of Western Blot analysis of lysates of cerebellum cultures containing all cell types of WT and NG2-KO mice with the specific OMI-inhibitor UCF101 (grey columns) or without (black columns). Cultures were exposed to H 2 O 2 for 18 hours. OPC were quantified by blotting for NG2 (WT), EYFP (KO) or JAM-A (for WT and KO). Degradation of PARP is an indication of cell death. In the presence of the OMI inhibitor UCF101, less death of NG2-lacking (NG2-KO) OPC is observed. The WB signals of samples from six individual animals (3WT; 3NG2-KO) with the indicated antibodies are shown on the right from which the quantitative data (bar graphs) is derived. All signals are normalised to Tubulin and compared to the unstressed controls (set as 100%) (n = 3, SEM, unpaired Student’s t-Test: p-value **<0.01; *<0.05). B) Cerebellum cultures of wildtype (WT) mice and NG2/EYFP knockout mice (NG2-KO) were incubated with 800μm H 2 O 2 for 18 hours, lysed and compared to unstressed controls by WB with cell-type-specific markers. Blots are shown of one experiment with three replicates. C) Quantification of WB signals for different cell-type specific markers against Tubulin of the cerebellum cultures, compared to unstressed cultures (EYFP and NG2 respectively for OPC, ASPA for oligodendrocytes, δ-Enolase for neurons and GFAP for astrocytes; (n = 4, SEM, unpaired Student’s t-Test: p-value *<0.05) as shown in the example (B). D) WB analyses with quantification of activated caspase-3 (cleaved caspase-3) of WT and NG2-KO cerebellum cultures with and without oxidative stress for 5 hours (n = 1). An example of apoptotic cell death in OPC in the cerebellum cultures from the NG2-KO mice after 5 hours of stress with 800μm H 2 O 2 stained with cleaved-caspase-3 (red), EYFP (green) and DAPI (blue) is shown. Scale bar = 50μm.

Article Snippet: The siRNA against NG2 (target sequence in the UTR1) and the control-siRNA (target sequence: 5′-AAT TCT CCG AAC GTG TCA CGT-3′ ) were obtained from QIAGEN.

Techniques: Western Blot, Derivative Assay, Knock-Out, Incubation, Staining

A) Diagram showing the domain organization of the NG2del proteins. The FLAG Tag was used in transfected cells in the MTT-assay, the HIS-Tag was utilised for purification of NG2del proteins for the protease-assay. TM indicates the transmembrane domain; the arrow indicates the position of the deletion (amino acids 478–2164) in NG2del. The amino acids QYWV represent the PDZ binding motif in the NG2del+ construct. B) MTT-assay of HEK293T cells transfected with an NG2-FLAG construct with an intact PDZ-binding-motif, which interacts with OMI/HtrA2 (NG2del+) and a NG2 construct in which the PDZ-binding-motif was deleted (NG2del-). The reduced MTT conversion with stress for 4h is compared to unstressed controls. HEK293T cells that express NG2del+ showed a higher percentage of viable cells under oxidative stress (n = 3, SEM, paired Student’s t-Test: p-value *<0.05). WB analysis with FLAG antibody (insert) shows similar expression of each transfected construct using the same amount of cells and plasmids. C) Scheme of the protease assay: to test the influence of NG2 on the OMI/HtrA2 protease activity, purified NG2del-HIS proteins (NG2del- without PDZ-binding motif and NG2del+ with the motif) were incubated with OMI/HtrA2-FLAG immunoprecipitates, obtained from HEK cells transfected with FLAG-tagged OMI. These were then incubated with the OMI/HtrA2 substrate β-casein for 5h at 37°C, to analyse whether binding of NG2 influenced the activity of the OMI serine protease. WB analysis (panel I) shows that equal amounts of the NG2del fusion proteins are used for each assay, as a control untransfected HEK lysate was used. D) WB analysis of the IPs used for the casein digestion shows that equal amounts of FLAG-tagged OMI/HtrA2 are used for each assay and that only NG2del+ (with the intact PDZ-binding motif) is co-immunoprecipitated in the OMI/HtrA2-FLAG IP. E) The degradation of β-casein in the supernatant from the same three experiments shown in D was analysed by gel electrophoresis and silver staining. Undigested casein is shown after incubation with the complex of OMI/HtrA2 and NG2 (NG2del+ or NG2del-). The buffer from the purification of the NG2del proteins was used as a control in D and E. Quantification of the signals of the casein band (NG2del+ and NG2del-) was normalized against the buffer control (left bar graphs). When the OMI/HtrA2 protease is inhibited by the interaction with the NG2del+ protein less casein is digested resulting in stronger signals (n = 3, SEM, unpaired Student’s t-Test: p-value *<0.05). In panel II it is shown that the OMI/HtrA2 protease inhibitor UCF101 largely prevents digestion of β-casein in this assay.

Journal: PLoS ONE

Article Title: The NG2 Proteoglycan Protects Oligodendrocyte Precursor Cells against Oxidative Stress via Interaction with OMI/HtrA2

doi: 10.1371/journal.pone.0137311

Figure Lengend Snippet: A) Diagram showing the domain organization of the NG2del proteins. The FLAG Tag was used in transfected cells in the MTT-assay, the HIS-Tag was utilised for purification of NG2del proteins for the protease-assay. TM indicates the transmembrane domain; the arrow indicates the position of the deletion (amino acids 478–2164) in NG2del. The amino acids QYWV represent the PDZ binding motif in the NG2del+ construct. B) MTT-assay of HEK293T cells transfected with an NG2-FLAG construct with an intact PDZ-binding-motif, which interacts with OMI/HtrA2 (NG2del+) and a NG2 construct in which the PDZ-binding-motif was deleted (NG2del-). The reduced MTT conversion with stress for 4h is compared to unstressed controls. HEK293T cells that express NG2del+ showed a higher percentage of viable cells under oxidative stress (n = 3, SEM, paired Student’s t-Test: p-value *<0.05). WB analysis with FLAG antibody (insert) shows similar expression of each transfected construct using the same amount of cells and plasmids. C) Scheme of the protease assay: to test the influence of NG2 on the OMI/HtrA2 protease activity, purified NG2del-HIS proteins (NG2del- without PDZ-binding motif and NG2del+ with the motif) were incubated with OMI/HtrA2-FLAG immunoprecipitates, obtained from HEK cells transfected with FLAG-tagged OMI. These were then incubated with the OMI/HtrA2 substrate β-casein for 5h at 37°C, to analyse whether binding of NG2 influenced the activity of the OMI serine protease. WB analysis (panel I) shows that equal amounts of the NG2del fusion proteins are used for each assay, as a control untransfected HEK lysate was used. D) WB analysis of the IPs used for the casein digestion shows that equal amounts of FLAG-tagged OMI/HtrA2 are used for each assay and that only NG2del+ (with the intact PDZ-binding motif) is co-immunoprecipitated in the OMI/HtrA2-FLAG IP. E) The degradation of β-casein in the supernatant from the same three experiments shown in D was analysed by gel electrophoresis and silver staining. Undigested casein is shown after incubation with the complex of OMI/HtrA2 and NG2 (NG2del+ or NG2del-). The buffer from the purification of the NG2del proteins was used as a control in D and E. Quantification of the signals of the casein band (NG2del+ and NG2del-) was normalized against the buffer control (left bar graphs). When the OMI/HtrA2 protease is inhibited by the interaction with the NG2del+ protein less casein is digested resulting in stronger signals (n = 3, SEM, unpaired Student’s t-Test: p-value *<0.05). In panel II it is shown that the OMI/HtrA2 protease inhibitor UCF101 largely prevents digestion of β-casein in this assay.

Article Snippet: The siRNA against NG2 (target sequence in the UTR1) and the control-siRNA (target sequence: 5′-AAT TCT CCG AAC GTG TCA CGT-3′ ) were obtained from QIAGEN.

Techniques: FLAG-tag, Transfection, MTT Assay, Purification, Protease Assay, Binding Assay, Construct, Expressing, Activity Assay, Incubation, Control, Immunoprecipitation, Nucleic Acid Electrophoresis, Silver Staining, Protease Inhibitor

A) MTT-assay with NG2-positive GBM cells (line R10) transfected with NG2siRNA or control siRNA. MTT conversion is displayed in comparison to unstressed controls, which are set as 100% (n = 4, SEM, unpaired Student’s t-Test: p-values **<0.01; *<0.05). LDH release is displayed in comparison to unstressed control (100%) (three replicates, SEM, unpaired Student’s t-Test: p-values **<0.01; *<0.05). WB analysis of the efficiency of NG2 knockdown in GBM cells is shown in the insert. B) MTT-assay and LDH-assay with GBM cells (R10) transfected with NG2siRNA and control siRNA and stressed with ONOO - (peroxynitrite). MTT conversion and LDH release are displayed in comparison to unstressed controls, which are set as 100% (n = 3, SEM, unpaired Student’s t-test: p-values *<0.05) are shown. C) Western-Blot analysis of PARP levels normalised against Tubulin in cells with and without NG2 knockdown. PARP levels of unstressed controls are set as 100% (n = 4, SEM, Student’s t-Test: p-value *<0.05) and an example of WB-analysis of PARP levels from one single experiment is shown. D) MTT-assay with NG2-positive GBM cells transfected with NG2siRNA or control siRNA and incubated with or without the OMI inhibitor UCF101. MTT conversion is displayed in comparison to unstressed controls, which are set as 100% (4 replicates, SEM, unpaired Student’s t-Test: p-values *<0.05).

Journal: PLoS ONE

Article Title: The NG2 Proteoglycan Protects Oligodendrocyte Precursor Cells against Oxidative Stress via Interaction with OMI/HtrA2

doi: 10.1371/journal.pone.0137311

Figure Lengend Snippet: A) MTT-assay with NG2-positive GBM cells (line R10) transfected with NG2siRNA or control siRNA. MTT conversion is displayed in comparison to unstressed controls, which are set as 100% (n = 4, SEM, unpaired Student’s t-Test: p-values **<0.01; *<0.05). LDH release is displayed in comparison to unstressed control (100%) (three replicates, SEM, unpaired Student’s t-Test: p-values **<0.01; *<0.05). WB analysis of the efficiency of NG2 knockdown in GBM cells is shown in the insert. B) MTT-assay and LDH-assay with GBM cells (R10) transfected with NG2siRNA and control siRNA and stressed with ONOO - (peroxynitrite). MTT conversion and LDH release are displayed in comparison to unstressed controls, which are set as 100% (n = 3, SEM, unpaired Student’s t-test: p-values *<0.05) are shown. C) Western-Blot analysis of PARP levels normalised against Tubulin in cells with and without NG2 knockdown. PARP levels of unstressed controls are set as 100% (n = 4, SEM, Student’s t-Test: p-value *<0.05) and an example of WB-analysis of PARP levels from one single experiment is shown. D) MTT-assay with NG2-positive GBM cells transfected with NG2siRNA or control siRNA and incubated with or without the OMI inhibitor UCF101. MTT conversion is displayed in comparison to unstressed controls, which are set as 100% (4 replicates, SEM, unpaired Student’s t-Test: p-values *<0.05).

Article Snippet: The siRNA against NG2 (target sequence in the UTR1) and the control-siRNA (target sequence: 5′-AAT TCT CCG AAC GTG TCA CGT-3′ ) were obtained from QIAGEN.

Techniques: MTT Assay, Transfection, Control, Comparison, Knockdown, Lactate Dehydrogenase Assay, Western Blot, Incubation

Review

Structured Review

Images

Similar Products

Image Search Results