Journal: The Journal of Neuroscience

Article Title: A Multilevel Functional Study of a SNAP25 At-Risk Variant for Bipolar Disorder and Schizophrenia

doi: 10.1523/JNEUROSCI.1040-17.2017

Figure Lengend Snippet: In vitro analysis of rs6039769 in N2a cells. Quantification of the SNAP25 expression level was estimated using a luciferase reporter gene under control of the human SNAP25 promoter region containing either the C or the A allele of rs6039769. Both constructs showed a higher reporter expression level than the empty vector. The C allele showed a 20% increase of the reporter expression level than the A allele. The values are reported as the mean ± SD. ***p < 0.0001.

Article Snippet: Functional in vitro study The Neuro-2a (N2a) mouse neuroblastoma cell line (catalog #400394/p451_Neuro-2A, CLS; RRID: CVCL_0470 ) was grown at 37°C, with 5% CO 2 in DMEM containing GlutaMAX (Thermo Fisher Scientific) and was supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: In Vitro, Expressing, Luciferase, Control, Construct, Plasmid Preparation

Journal: Journal of Nanophotonics

Article Title: Mouse neuroblastoma cell-based model and the effect of epileptic events on calcium oscillations and neural spikes

doi: 10.1117/1.jnp.7.073794

Figure Lengend Snippet: Fig. 2 Fluorescent imaging of mouse neuroblastoma neuro2a (N2A) cells using fluo-4 NW: (a) control cells; (b) 30 min after the injection of 4-AP (10 mM); and (c) 30 min after the injection of 4-AP (50 mM).

Article Snippet: 2.1 Mouse Neuroblastoma N2A Cell Culture The mouse neuroblastoma N2A cells were purchased from American Type Culture Collection (CCl-131, Manassas, Virginia) and cultured at a density of 1.0 × 104 in Dulbecco’s modified Eagle’s medium (DMEM) that was supplemented with heat-inactivated 10% fetal bovine Journal of Nanophotonics 073794-2 Vol.

Techniques: Imaging, Control, Injection

Journal: Journal of Nanophotonics

Article Title: Mouse neuroblastoma cell-based model and the effect of epileptic events on calcium oscillations and neural spikes

doi: 10.1117/1.jnp.7.073794

Figure Lengend Snippet: Fig. 3 Calcium oscillations in mouse neuroblastoma N2A cells: control cells (blue line), 30 min after the injection of the 4-AP with 10 mM (black line) and 50 mM (red line).

Article Snippet: 2.1 Mouse Neuroblastoma N2A Cell Culture The mouse neuroblastoma N2A cells were purchased from American Type Culture Collection (CCl-131, Manassas, Virginia) and cultured at a density of 1.0 × 104 in Dulbecco’s modified Eagle’s medium (DMEM) that was supplemented with heat-inactivated 10% fetal bovine Journal of Nanophotonics 073794-2 Vol.

Techniques: Control, Injection

Journal: Molecular cell

Article Title: The Kelch repeat protein KLHDC10 regulates oxidative stress-induced ASK1 activation by suppressing PP5.

doi: 10.1016/j.molcel.2012.09.018

Figure Lengend Snippet: Figure 4. The CRL2 Complex Is Dispensable for ASK1 Activation by Slim (A) ClustalW alignment of the amino acid sequences of the C-terminal regions of Slim, KLHDC10, and KLHDC2. Slim and KLHDC10 contain BC box- and Cul2 box-like sequences. f indicates hydrophobic amino acids. (B) Schematic representation of human KLHDC10 WT and its mutants DBC, AP, and DN and a splicing variant of mouse KLHDC10, DEx2, as well as Slim WT and DBC. (C) HEK293 cells were transiently transfected with the indicated constructs. After 24 hr, cells were lysed and subjected to immunoprecipitation (IP) with anti-Flag antibody followed by immunoblotting (IB). (D) Neuro2A cells were transiently transfected with the indicated constructs. After 24 hr, cells were lysed and subjected to IP with anti-Flag antibody followed by IB. (E) S2 cells were transiently transfected with the indicated constructs and pWAGAL4. After 24 hr, the cells were lysed and subjected to IB. (F) HEK293 cells were transiently transfected with the indicated constructs. After 24 hr, the cells were lysed and subjected to IB.

Article Snippet: For IP of endogenous KLHDC10, Neuro2A cell lysates were incubated with control rat IgG (Santa Cruz) or KLHDC10 antibody (clone S-20, Supplemental Experimental Procedures) and immunoprecipitated with protein G Sepharose (GE Healthcare).

Techniques: Activation Assay, Variant Assay, Transfection, Construct, Immunoprecipitation, Western Blot

Journal: Molecular cell

Article Title: The Kelch repeat protein KLHDC10 regulates oxidative stress-induced ASK1 activation by suppressing PP5.

doi: 10.1016/j.molcel.2012.09.018

Figure Lengend Snippet: Figure 6. H2O2-Dependent Interaction of KLHDC10 with PP5 (A and B) HEK293 cells were transiently transfected with the indicated constructs. After 48 hr, the cells were treated with 5 mM H2O2 for the indicated periods and then lysed and subjected to immunoprecipitation (IP) with anti-Flag antibody followed by immunoblotting (IB). (C) Neuro2A cells were transfected with a negative control siRNA or an siRNA that targets Cul2. After 48 hr, the cells were treated with 1 mM H2O2 for 1 hr and then lysed and subjected to IP with control IgG or KLHDC10 antibody followed by IB. The asterisk indicates the bands derived from immunoglobulin heavy chain or nonspecific bands. The total amount of PP5 (full length and cleaved) coimunoprecipitated with KLHDC10 was quantified and was shown as the fold changes.

Article Snippet: For IP of endogenous KLHDC10, Neuro2A cell lysates were incubated with control rat IgG (Santa Cruz) or KLHDC10 antibody (clone S-20, Supplemental Experimental Procedures) and immunoprecipitated with protein G Sepharose (GE Healthcare).

Techniques: Transfection, Construct, Immunoprecipitation, Western Blot, Negative Control, Control, Derivative Assay

Journal: Molecular cell

Article Title: The Kelch repeat protein KLHDC10 regulates oxidative stress-induced ASK1 activation by suppressing PP5.

doi: 10.1016/j.molcel.2012.09.018

Figure Lengend Snippet: Figure 7. KLHDC10 Is Required for H2O2- Induced Activation of ASK1 and Cell Death (A and B) Neuro2A cells were transfected with a negative control siRNA or an siRNA that targets KLHDC10. After 48 hr, the cells were treated with 0.5 mM H2O2 for the indicated periods, then lysed and subjected to immunoblotting (IB) with the indicated antibodies (A), or after 43 hr, the cells were treated with 1 mM H2O2 for 5 hr and then analyzed with an LDH assay (B). The results shown are the means of three independent ex- periments. Error bars indicate SEM (**p < 0.003, ***p < 0.007, Student’s t test). (C and D) Neuro2A cells were transfected with the indicated siRNAs. After 48 hr, the cells were har- vested and replated. After 24 hr, the indicated siRNAs were transfected again. After 48 hr, the cells were treated with 0.5 mM H2O2 for the indi- cated periods and then lysed and subjected to IB (C), or after 43 hr, the cells were treated with 1 mM H2O2 for 5 hr and then analyzed with an LDH assay (D). The results shown are the means of at least five independent experiments. Error bars indicate SEM (*p < 0.05, Student’s t test). The asterisk indicates nonspecific bands.

Article Snippet: For IP of endogenous KLHDC10, Neuro2A cell lysates were incubated with control rat IgG (Santa Cruz) or KLHDC10 antibody (clone S-20, Supplemental Experimental Procedures) and immunoprecipitated with protein G Sepharose (GE Healthcare).

Techniques: Activation Assay, Transfection, Negative Control, Western Blot, Lactate Dehydrogenase Assay

Journal: International Journal of Molecular Sciences

Article Title: Molecular Insights of Neuroprotective Effect of Cornulaca monacantha Extract Against LPS-Induced Neuroinflammation Supported by Metabolic Profiling and Protein Interaction Analysis

doi: 10.3390/ijms27052263

Figure Lengend Snippet: Effect of C. monacantha methanolic extract on Neuro-2a cell viability. The tested concentrations of the methanolic extract ranged from 31.25 to 1000 µg/mL. Data are expressed as percentage of the untreated control (set at 100%) and presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc multiple-comparison test ( p < 0.05). a: significant vs. control; b: significant vs. 31.25 µg/mL; c: significant vs. 62.5 µg/mL; d: significant vs. 125 µg/mL.

Article Snippet: In this study, the Neuro-2a (Cat. # ABC-TC0819) mouse neuroblastoma cell line (AcceGen Biotech, Fairfield, NJ, USA) was used.

Techniques: Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Molecular Insights of Neuroprotective Effect of Cornulaca monacantha Extract Against LPS-Induced Neuroinflammation Supported by Metabolic Profiling and Protein Interaction Analysis

doi: 10.3390/ijms27052263

Figure Lengend Snippet: Effect of C. monacantha extract on LPS-induced neuroinflammation in Neuro-2a cells. ( a ) IL-1β and ( b ) MCP-1. Data are expressed as mean ± SD. * p < 0.05. LPS: lipopolysaccharide; IL-1β: interleukin-1β; MCP-1: monocyte chemoattractant protein-1.

Article Snippet: In this study, the Neuro-2a (Cat. # ABC-TC0819) mouse neuroblastoma cell line (AcceGen Biotech, Fairfield, NJ, USA) was used.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Molecular Insights of Neuroprotective Effect of Cornulaca monacantha Extract Against LPS-Induced Neuroinflammation Supported by Metabolic Profiling and Protein Interaction Analysis

doi: 10.3390/ijms27052263

Figure Lengend Snippet: Effect of C. monacantha extract on the mRNA expression of the Nrf2 pathway and related genes in Neuro-2a cells. ( a ) Nrf2, ( b ) NF-κB, ( c ) Hmox1, and ( d ) NQO-1. Data are expressed as mean ± SD. * p < 0.05. LPS: lipopolysaccharide; Nrf2: nuclear factor erythroid 2–related factor 2; NF-κB: nuclear factor kappa B; Hmox1: heme oxygenase 1; NQO-1: NAD(P)H quinone dehydrogenase 1.

Article Snippet: In this study, the Neuro-2a (Cat. # ABC-TC0819) mouse neuroblastoma cell line (AcceGen Biotech, Fairfield, NJ, USA) was used.

Techniques: Expressing

Journal: Advanced Science

Article Title: A Bioinspired Manganese‐Organic Framework Ameliorates Ischemic Stroke through its Intrinsic Nanozyme Activity and Upregulating Endogenous Antioxidant Enzymes

doi: 10.1002/advs.202206854

Figure Lengend Snippet: The effect of pDA‐MNOF on upregulating HO1 and SOD2 via STAT3 signaling. a) Western blot analysis of p‐STAT3, STAT3, HO1, and SOD2 expression in N2a cells treated with 12.5 µg mL −1 pDA‐MNOF for various time durations. b) Quantification of the band intensity ratios of p‐STAT3/STAT3, HO1/ β ‐actin, and SOD2/ β ‐actin in (a). t‐STAT3 indicated total proteins of p‐STAT3 and STAT3. c) Quantification of fluorescent intensity of p‐STAT3, HO1, and SOD2 in PBS or 12.5 µg mL −1 pDA‐MNOF‐treated N2a cells ( n = 4). Data were presented with mean ± s.d.; *, p < 0.05; **, p < 0.01; T ‐test. d) Representative images of immunofluorescent staining for p‐STAT3, HO‐1, and SOD‐2 (red) in indicated groups. Nuclei were stained in blue, and cell body was outlined with white dashed lines. Scale bar, 20 µm. e) Western blot analysis of p‐STAT3, STAT3, HO1, and SOD2 expression in N2a cells treated with different concentrations of pDA‐MNOF for 12 h. f) Quantification of the band intensity ratios of p‐STAT3/STAT3, HO1/ β ‐actin, and SOD2/ β ‐actin in (e). g) Experimental scheme for the RNA interference and pathway inhibition assays. h) Western blot analysis of p‐STAT3 and STAT3 in N2a cells treated with PBS, siRNA Control (siRNA Ctrl), siRNA 1#, siRNA 2#, and siRNA 3#. i) Quantification of the band intensity ratios of p‐STAT3/ β ‐actin and STAT3/ β ‐actin in (h). j) N2a cells were transfected with siRNA Ctrl, siRNA 2#, and siRNA 3#, followed by treatment with pDA‐MNOF; the lysates were analyzed with indicated antibodies. k) Quantification of the band intensity ratios of p‐STAT3/ β ‐actin, HO1/ β ‐actin, and SOD2/ β ‐actin in (j). Data were presented with mean ± s.d.; *, p < 0.05; ANOVA. l) N2a cells were treated with PBS or pDA‐MNOF, followed by treatment with AG490; the lysates were analyzed with indicated antibodies. m) Quantification of the band intensity ratios of p‐STAT3/STAT3, HO1/ β ‐actin, and SOD2/ β ‐actin in (l) ( n = 3). Data were presented with mean ± s.d.; *, p < 0.05; **, p < 0.01; ANOVA.

Article Snippet: To assess the protein levels of VEGF, the supernatant of N2a cells that were treated with PBS or various concentrations of pDA‐MNOF for 24 h was collected for ELISA detection (Elabscience, China).

Techniques: Western Blot, Expressing, Staining, Inhibition, Control, Transfection

Journal: Advanced Science

Article Title: A Bioinspired Manganese‐Organic Framework Ameliorates Ischemic Stroke through its Intrinsic Nanozyme Activity and Upregulating Endogenous Antioxidant Enzymes

doi: 10.1002/advs.202206854

Figure Lengend Snippet: pDA‐MNOF protects neuronal cells against ROS‐induced injury. a) DCFH‐DA staining for detecting ROS in the PBS or pDA‐MNOF‐treated N2a cells after being treated with an 8 h OGD. N2a cells cultured in regular condition were set as the control. Scale bars, 50 µm. b) Quantification of percentages of DCFH‐DA positive (DCFDA + ) cells in indicated groups in (a) ( n = 5). Data were presented with mean ± s.d.; *, p < 0.05; ****, p < 0.0001; ANOVA. c) Flow cytometric analysis of DCFH‐DA positive cell proportions in the three groups. d) Fluorescence intensity detection in cell lysis of DCFH‐DA‐stained N2a cells in indicated groups ( n = 5). Data were presented with mean ± s.d.; *, p < 0.05; ***, p < 0.001; ANOVA. e) Schematic illustration of determining the contribution ratios of two effects of pDA‐MNOF on scavenging ROS. Briefly, N2a cells were treated with PBS, or 12.5 µg mL −1 pDA‐MNOF for 12 h, and their SOD‐like activities were determined as A 0 and A 1 , respectively. Cell lysis of pDA‐MNOF‐treated N2a cells was subject to ultracentrifugation for removing the residual pDA‐MNOF, and the SOD‐like activity was determined as A 3 . f) Quantification of contribution ratios of two effects of pDA‐MNOF on scavenging ROS. The formula was listed to the right. g) Live & Dead staining for PBS or pDA‐MNOF‐treated N2a cells after an 8 h OGD followed by a 16 h regular incubation. Scale bars, 100 µm. h) Quantification of percentages of dying cells in indicated groups in (g) ( n = 5). Data were presented with mean ± s.d.; ****, p < 0.0001; ANOVA. i) Cell viability of N2a cells in indicated groups in CCK‐8 assay ( n = 5). Data were presented with mean ± s.d.; **, p < 0.01; ****, p < 0.0001; ANOVA. j) Schematics of pDA‐MNOF utilizing nanozyme activity and upregulating endogenous HO1/SOD2, jointly scavenging ROS.

Article Snippet: To assess the protein levels of VEGF, the supernatant of N2a cells that were treated with PBS or various concentrations of pDA‐MNOF for 24 h was collected for ELISA detection (Elabscience, China).

Techniques: Staining, Cell Culture, Control, Fluorescence, Lysis, Activity Assay, Incubation, CCK-8 Assay

Journal: Advanced Science

Article Title: A Bioinspired Manganese‐Organic Framework Ameliorates Ischemic Stroke through its Intrinsic Nanozyme Activity and Upregulating Endogenous Antioxidant Enzymes

doi: 10.1002/advs.202206854

Figure Lengend Snippet: pDA‐MNOF promotes angiogenesis. a) Schematics showing VEGF, a downstream gene of STAT3 signaling, was activated by pDA‐MNOF. b) The mRNA levels of VEGF in N2a cells treated with PBS (control) or pDA‐MNOF ( n = 3). Data were presented with mean ± s.d.; **, p < 0.01; ****, p < 0.0001; ANOVA. c) The levels of VEGF protein secreted from the PBS (control) or pDA‐MNOF‐treated N2a cells ( n = 3). Data were presented with mean ± s.d.; ***, p < 0.001; ****, p < 0.0001; ANOVA. d) Capillary‐like tube formation assay performed on C166 cells (murine endothelial cell line) that were cultured with regular N2a culture medium or conditioned medium obtained from the pDA‐MNOF‐treated N2a cells. 5pDA‐MNOF, 12.5pDA‐MNOF, or 25pDA‐MNOF represent conditioned medium that was from the 5, 12.5, or 25 µg mL −1 pDA‐MNOF‐treated N2a cells, respectively. The branch points were indicated by red arrowheads, and the structure between two adjacent arrowheads was a branch. e) Quantification of branch point numbers, tube length, and tube numbers in capillary‐like tube formation assay in (d) ( n = 5 repeats). f) Schematics of cerebral ventricle injection of PBS and pDA‐MNOF for further evaluating in vivo angiogenesis. g) Relative VEGF mRNA levels in brain tissue of the MCAo mice receiving cerebral ventricle injection of PBS or pDA‐MNOF ( n = 9). The mice received sham operation were set as control ( n = 3). Data were presented with mean ± s.d.; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ANOVA. h) Representative fluorescence images of vessels (CD31, red) and astrocytes (GFAP, green) in the infarct (an asterisk) and peri‐infarct 4 weeks after the mice received cerebral ventricle injection of PBS or pDA‐MNOF. The boundary between the infarct and peri‐infarct was indicated by white dashed lines. i,j) Quantification of percentages of CD31 positive (CD31 + ) area in i) the infarct and j) peri‐infarct, respectively ( n = 9). Data were presented with mean ± s.d.; *, p < 0.05; **, p < 0.01; ANOVA.

Article Snippet: To assess the protein levels of VEGF, the supernatant of N2a cells that were treated with PBS or various concentrations of pDA‐MNOF for 24 h was collected for ELISA detection (Elabscience, China).

Techniques: Control, Capillary Tube Formation Assay, Cell Culture, Injection, In Vivo, Fluorescence

Journal: iScience

Article Title: Drp1 SUMO/deSUMOylation by Senp5 isoforms influences ER tubulation and mitochondrial dynamics to regulate brain development

doi: 10.1016/j.isci.2021.103484

Figure Lengend Snippet:

Article Snippet: Neuro2a cells , JCRB , Cat#IFO50081.

Techniques: Recombinant, Blocking Assay, cDNA Synthesis, Plasmid Preparation, Western Blot, Cell Culture, Mutagenesis, Software