nsc34 cell line  (Procell Inc)

Journal: Molecular Medicine

Article Title: The therapeutic potential of Apigenin in amyotrophic lateral sclerosis through ALDH1A2/Nrf2/ARE signaling

doi: 10.1186/s10020-024-00977-7

Figure Lengend Snippet: Effect of APG on viability and apoptosis in NSC34 neuronal cells. A CCK8 assay to evaluate the effects of different doses of APG on the viability of NSC34 cells in vitro. B Flow cytometry analysis of apoptosis in NSC34 cells harboring the SOD1*G93A mutation treated with APG. C Western blot analysis of apoptosis-related proteins (Bcl-2, caspase-3, Bax) in SOD1*G93A mutant NSC34 cells treated with APG. D Flow cytometry assessment of ROS levels in SOD1*G93A mutant NSC34 cells under APG treatment. E - H ELISA for quantification of OS markers (MDA, 4-HNE, SOD, ALDH) in SOD1*G93A mutant NSC34 cells treated with APG. I qRT-PCR analysis of ALDH1A2 mRNA levels, and (J) Western blot analysis of ALDH1A2 protein levels in APG-treated SOD1*G93A mutant NSC34 cells. * P < 0.05, ** P < 0.01, * P < 0.001

Article Snippet: 293FT and NSC34 cell lines from Procell (Wuhan, China) were cultured in DMEM containing 10% fetal bovine serum (FBS) at 37 °C.

Techniques: CCK-8 Assay, In Vitro, Flow Cytometry, Mutagenesis, Western Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Molecular Medicine

Article Title: The therapeutic potential of Apigenin in amyotrophic lateral sclerosis through ALDH1A2/Nrf2/ARE signaling

doi: 10.1186/s10020-024-00977-7

Figure Lengend Snippet: Effects of ALDH1A2 knockdown and APG treatment on ALS model and OS markers. A - B Validation and selection of the most effective sh-ALDH1A2 to suppress ALDH1A2 expression by qRT-PCR and Western blot analysis. C Flow cytometry to evaluate apoptosis in NSC34 cells treated with APG and sh-ALDH1A2. D Western blot to evaluate the levels of apoptosis-related proteins (Bcl-2, caspase-3, Bax) in NSC34 cells treated with both APG and sh-ALDH1A2. E Flow cytometric analysis of ROS levels in treated NSC34 cells. F - I ELISA for quantification of OS markers (MDA, 4-HNE, SOD, ALDH) in NSC34 cells treated with APG and sh-ALDH1A2. J Western blot analysis of ALDH1A2 protein levels. K - L qRT-PCR and Western blot analysis of Nrf2, HO-1, NQO1 mRNA and protein levels in NSC34 cells treated with APG and sh-ALDH1A2. * P < 0.05, ** P < 0.01, * P < 0.001

Article Snippet: 293FT and NSC34 cell lines from Procell (Wuhan, China) were cultured in DMEM containing 10% fetal bovine serum (FBS) at 37 °C.

Techniques: Knockdown, Biomarker Discovery, Selection, Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Specific Post-Translational Modifications of VDAC3 in ALS-SOD1 Model Cells Identified by High-Resolution Mass Spectrometry

doi: 10.3390/ijms232415853

Figure Lengend Snippet: Sequence coverage map of VDAC3 from NSC34, NSC34-SOD1WT and NSC34-SOD1G93A cell lines obtained by tryptic and chymotryptic digestion. The solid line indicates the sequence that was obtained from tryptic peptides; dotted lines: sequence obtained from chymotryptic peptides. Unique tryptic (indicated in bold and blue) and chymotryptic (indicated in bold and black) peptides originating from missed cleavages were used to distinguish and cover the sequences shared by isoforms. Sequences shared by multiple isoforms are shown in red. Sequence numbering considered the starting methionine residue, which is eliminated during protein maturation.

Article Snippet: NSC34 cell lines (mouse motor neuron-like) obtained from CELLutions Biosystem Inc. were stably transfected with pTet-ON plasmid (Clontech, Mountain View, CA, USA) harboring sequences encoding human SOD1 wt (NSC34-SOD1WT) or G93A (NSC34-SOD1G93A) and used as non-ALS motor neuron line and ALS motor neuron line, respectively [ ].

Techniques: Sequencing, Residue

Journal: International Journal of Molecular Sciences

Article Title: Specific Post-Translational Modifications of VDAC3 in ALS-SOD1 Model Cells Identified by High-Resolution Mass Spectrometry

doi: 10.3390/ijms232415853

Figure Lengend Snippet: Ox/Red ratio (average and standard deviation) of the absolute intensities of the molecular ions of sulfur containing tryptic peptides found in the analysis of VDAC3 from NSC34, NSC34-SOD1WT, NSC34-SOD1G93A cell lines reduced with DTT, carboxyamidomethylated and digested in-solution.

Article Snippet: NSC34 cell lines (mouse motor neuron-like) obtained from CELLutions Biosystem Inc. were stably transfected with pTet-ON plasmid (Clontech, Mountain View, CA, USA) harboring sequences encoding human SOD1 wt (NSC34-SOD1WT) or G93A (NSC34-SOD1G93A) and used as non-ALS motor neuron line and ALS motor neuron line, respectively [ ].

Techniques: Standard Deviation, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Specific Post-Translational Modifications of VDAC3 in ALS-SOD1 Model Cells Identified by High-Resolution Mass Spectrometry

doi: 10.3390/ijms232415853

Figure Lengend Snippet: Ox/Red ratio (average and standard deviation) of the absolute intensities of the molecular ions of sulfur containing chymotryptic peptides found in the analysis of VDAC3 from NSC34, NSC34-SOD1WT, NSC34-SOD1G93A cell lines reduced with DTT, carboxyamidomethylated and digested in-solution.

Article Snippet: NSC34 cell lines (mouse motor neuron-like) obtained from CELLutions Biosystem Inc. were stably transfected with pTet-ON plasmid (Clontech, Mountain View, CA, USA) harboring sequences encoding human SOD1 wt (NSC34-SOD1WT) or G93A (NSC34-SOD1G93A) and used as non-ALS motor neuron line and ALS motor neuron line, respectively [ ].

Techniques: Standard Deviation, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Specific Post-Translational Modifications of VDAC3 in ALS-SOD1 Model Cells Identified by High-Resolution Mass Spectrometry

doi: 10.3390/ijms232415853

Figure Lengend Snippet: MS/MS spectrum of the doubly charged molecular ion at m/z 660.2664 (calculated 660.2663) of the N-terminal tryptic peptide of VDAC3 from NSC34-SOD1WT cell line with cysteine residue 2 in the form of sulfonic acid and cysteine residue 8 in the carboxyamidomethylated form. The inset shows the full scan mass spectrum of the molecular ion. Fragment ions originating from the neutral loss of H 2 O are indicated by an asterisk. The fragment ion originating from the neutral loss of NH 3 is indicated by two asterisks.

Article Snippet: NSC34 cell lines (mouse motor neuron-like) obtained from CELLutions Biosystem Inc. were stably transfected with pTet-ON plasmid (Clontech, Mountain View, CA, USA) harboring sequences encoding human SOD1 wt (NSC34-SOD1WT) or G93A (NSC34-SOD1G93A) and used as non-ALS motor neuron line and ALS motor neuron line, respectively [ ].

Techniques: Tandem Mass Spectroscopy, Residue

Journal: International Journal of Molecular Sciences

Article Title: Specific Post-Translational Modifications of VDAC3 in ALS-SOD1 Model Cells Identified by High-Resolution Mass Spectrometry

doi: 10.3390/ijms232415853

Figure Lengend Snippet: Ox/Red ratio (average and standard deviation) of the absolute intensities of the molecular ions of tryptic peptides containing succinated and non-succinated cysteines found in the analysis of VDAC3 from NSC34, NSC34-SOD1WT, NSC34-SOD1G93A cell lines reduced with DTT, carboxyamidomethylated and digested in-solution.

Article Snippet: NSC34 cell lines (mouse motor neuron-like) obtained from CELLutions Biosystem Inc. were stably transfected with pTet-ON plasmid (Clontech, Mountain View, CA, USA) harboring sequences encoding human SOD1 wt (NSC34-SOD1WT) or G93A (NSC34-SOD1G93A) and used as non-ALS motor neuron line and ALS motor neuron line, respectively [ ].

Techniques: Standard Deviation, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Specific Post-Translational Modifications of VDAC3 in ALS-SOD1 Model Cells Identified by High-Resolution Mass Spectrometry

doi: 10.3390/ijms232415853

Figure Lengend Snippet: Ox/Red ratio (average and standard deviation) of the absolute intensities of the molecular ions of tryptic peptides containing deamidated and non-deamidated asparagine found in the analysis of VDAC3 from NSC34-SOD1G93A cell line reduced with DTT, carboxyamidomethylated and digested in-solution.

Article Snippet: NSC34 cell lines (mouse motor neuron-like) obtained from CELLutions Biosystem Inc. were stably transfected with pTet-ON plasmid (Clontech, Mountain View, CA, USA) harboring sequences encoding human SOD1 wt (NSC34-SOD1WT) or G93A (NSC34-SOD1G93A) and used as non-ALS motor neuron line and ALS motor neuron line, respectively [ ].

Techniques: Standard Deviation, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Specific Post-Translational Modifications of VDAC3 in ALS-SOD1 Model Cells Identified by High-Resolution Mass Spectrometry

doi: 10.3390/ijms232415853

Figure Lengend Snippet: MS/MS spectrum of the doubly charged molecular ion at m/z 924.9586 (calculated 924.9582) of the VDAC3 tryptic peptide from NSC34-SOD1G93A cell line containing the asparagine residue 215 in the deamidated form. The inset shows the full scan mass spectrum of molecular ion. Fragment ions originated from the neutral loss of H 2 O are indicated by an asterisk. Fragment ions originated from the neutral loss of NH 3 are indicated by two asterisks.

Article Snippet: NSC34 cell lines (mouse motor neuron-like) obtained from CELLutions Biosystem Inc. were stably transfected with pTet-ON plasmid (Clontech, Mountain View, CA, USA) harboring sequences encoding human SOD1 wt (NSC34-SOD1WT) or G93A (NSC34-SOD1G93A) and used as non-ALS motor neuron line and ALS motor neuron line, respectively [ ].

Techniques: Tandem Mass Spectroscopy, Residue

Journal: International Journal of Molecular Sciences

Article Title: Specific Post-Translational Modifications of VDAC3 in ALS-SOD1 Model Cells Identified by High-Resolution Mass Spectrometry

doi: 10.3390/ijms232415853

Figure Lengend Snippet: Ox/Red ratio (average and standard deviation) of the absolute intensities of the molecular ions of tryptic peptides containing succinimide intermediate found in the analysis of VDAC3 from NSC34, NSC34-SOD1WT, NSC34-SOD1G93A cell lines reduced with DTT, carboxyamidomethylated and digested in-solution.

Article Snippet: NSC34 cell lines (mouse motor neuron-like) obtained from CELLutions Biosystem Inc. were stably transfected with pTet-ON plasmid (Clontech, Mountain View, CA, USA) harboring sequences encoding human SOD1 wt (NSC34-SOD1WT) or G93A (NSC34-SOD1G93A) and used as non-ALS motor neuron line and ALS motor neuron line, respectively [ ].

Techniques: Standard Deviation, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Specific Post-Translational Modifications of VDAC3 in ALS-SOD1 Model Cells Identified by High-Resolution Mass Spectrometry

doi: 10.3390/ijms232415853

Figure Lengend Snippet: Retention time, experimentally measured and calculated monoisotopic m/z of the molecular ions, position in the sequence, absolute intensities and peptide sequence of tryptic fragment containing asparagine residue 76 in succinimide intermediate form and asparagine residue 79 in isoaspartate methyl ester form found in the analysis of VDAC3 from NSC34 and NSC34-SOD1WT cell lines reduced with DTT, carboxyamidomethylated and digested in-solution.

Article Snippet: NSC34 cell lines (mouse motor neuron-like) obtained from CELLutions Biosystem Inc. were stably transfected with pTet-ON plasmid (Clontech, Mountain View, CA, USA) harboring sequences encoding human SOD1 wt (NSC34-SOD1WT) or G93A (NSC34-SOD1G93A) and used as non-ALS motor neuron line and ALS motor neuron line, respectively [ ].

Techniques: Sequencing, Residue

Journal: The EMBO Journal

Article Title: Autophagy regulates neuronal excitability by controlling cAMP /protein kinase A signaling at the synapse

doi: 10.15252/embj.2022110963

Figure Lengend Snippet: A Western Blot analysis of PKA R1α protein levels in NSC34 cells, treated with 250 nM Torin1 or deprived of amino‐acids and serum for 16 h. Lysosomal degradation was blocked in the last 4 h using 67 nM of Bafilomycin A (BafA). B Starvation significantly reduced PKA R1α protein levels in NSC34 cells (vehicle set to 100%, starvation: 31.19 ± 5.085%, P < 0.0001, one‐tailed unpaired t ‐test). n = 5. C Application of 67 nM BafA for 4 h before harvesting was sufficient to stabilize the PKA R1α protein levels in starved NSC34 cells (starvation set to 100%, starvation + BafA: 155.5 ± 13.93%, P = 0.002, one‐tailed unpaired t ‐test). n = 5. D, E 16 h starvation significantly increased intracellular cAMP level measured as mean intensity in MAP2‐positive cultured primary neurons (Vehicle: 212.4 ± 5.619, Starvation: 255.1 ± 5.715; P < 0.0001, two‐tailed paired t ‐test). n Vehicle = 314, n Starvation = 306 neurons from n = 3. F, G Phosphorylation state of proteins containing PKA substrate RRXS/T motif was increased upon 16 h of amino‐acid and serum starvation in primary cortico‐hippocampal neurons at DIV14 and was suppressed by 1 μM H89 supplementation in the media (Vehicle set to 100%, Starvation: 326.60 ± 94.53%, H89: 214.80 ± 56.87%, P = 0.033, one‐way ANOVA with Dunn's multiple comparison test). n = 4. The pPKA substrate levels were normalized to the total protein amount stained with Ponceau S. H, I Treatment of NSC34 cells with BafA (67 nM) for 6 h significantly increased PKA R1α protein levels compared to the DMSO‐treated group set to 100% (BafA: 198.9 ± 28.44%, P = 0.003, one‐tailed unpaired t ‐test). n = 6. J Western blot analysis of purified autophagosomes (AVs) (50 μg AVs/lane) with and without Proteinase K (PK) treatment. TX‐100 (1% final) was used as a positive control for the activity of PK. Synaptosome lysates (Syn, 30 μg/lane) were used as a positive control for the signal of antibodies. K, L Representative confocal images of the hippocampus CA1 area from Atg5 flox: CamKIIα ‐Cre (K) and Atg5 flox: Slc32a1 ‐Cre (L) KO mice, immunostained for PKA R1‐α/β and co‐immunostained for p62 and/or NBR1. White rectangular boxes indicate areas magnified to the right. White arrows indicate p62/NBR1 inclusion bodies positive for PKA R1. Scale bar: large panel 15 μm, small panel: 5 μm. Representative pictures for WT controls are shown in Appendix Fig . M, N Representative fluorescent images and subsequent analysis of PKA R1‐ α/p62 colocalization (Pearson's correlation coefficient) in primary cortico‐hippocampal neurons, which have undergone 16 h amino‐acids and serum starvation and were additionally treated with BafA (67 nM) to visualize the lysosomes (control: 0.68 ± 0.019, starvation: 0.58 ± 0.015, starvation + BafA: 0.83 ± 0.014; P control vs starvation < 0.0001, P control vs starvation+BafA < 0.0001, P starvation vs starvation+BafA < 0.0001 Ordinary one‐way ANOVA with Tukey's multiple comparison test). n control = 36, n starvation = 38, n starvation+BafA = 36 neurons from n = 3. Scale bar: 5 μm. O, P 3D‐reconstruction of Atg5 flox: CamKIIα ‐Cre KO soma (O), as well as the immunohistochemical profile and the 3D surface rendering of Atg5 flox: CamKIIα ‐Cre KO neuropil (P) revealing the PKA R1‐β/p62 colocalization in the soma (white arrows), but not in processes (yellow arrows). Scale bar: 10 μM. Q, R Co‐immunoprecipitation of endogenous PKA R1‐β with PKA‐Cα from Atg5 flox: CamKIIα ‐Cre WT/KO mouse brain lysates (WT set to 100%, KO: 170.3 ± 14.88%, P = 0.005, one‐tailed unpaired t ‐test, n = 3). Input, 1.5% of the total lysate was added to the assay. Data information: * P < 0.05; ** P < 0.01; *** P < 0.001. All data represent mean ± SEM. All n represent biological replicates. Source data are available online for this figure.

Article Snippet: Primary neurons, and NSC34 (CLU140, Cedarlane, provided by Prof. B. Wirth) cells were harvested in RIPA buffer containing Protease Inhibitor (Roche) and Phosphatase Inhibitor (ThermoScientific) using a cell scraper.

Techniques: Western Blot, One-tailed Test, Cell Culture, Two Tailed Test, Staining, Purification, Positive Control, Activity Assay, Immunohistochemical staining, Immunoprecipitation

Journal: Antioxidants

Article Title: Dipeptide Repeat Pathology in C9orf72 -ALS Is Associated with Redox, Mitochondrial and NRF2 Pathway Imbalance

doi: 10.3390/antiox11101897

Figure Lengend Snippet: Endogenous and exogenous exposure to poly-GR and poly-PR decrease mitochondrial membrane potential and induce cell death. NSC34 cells were either transiently transfected with EGFP (control), EGFP-GR 50 or EGFP-PR 50 during 48 h or treated with 6 µM of HA, HA-GR 20 or HA-PR 20 peptides for 6 h (immunofluorescence) or 24 h (flow cytometry and viability assay). ( A , D ) EGFP fluorescence or HA immunofluorescence staining was analyzed by microscopy. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 40 µm. ( B , E ) cells were treated with 0.5 µM MitoTracker Red FM during 30 min and fluorescence was measured by flow cytometry analysis. Each point depicts the geometric mean of probe fluorescence in cells from that sample (( B ), alive EGFP + cells, ( E ), all alive cells). ( C , F ) viability of the cells was measured using an MTT assay. Bar height indicates the mean of the whole group and error bars indicate the SEM. Statistical analysis was performed with one-way ANOVA. Dunnett’s post-hoc test. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The Mouse Motor Neuron-Like Hybrid Cell Line (NSC34) (Cedarlane Laboratories, Burlington, Canada) was grown in DMEM (Dulbecco’s modified Eagle’s medium with high glucose; Sigma-Aldrich, San Luis, MO, USA; ID D5648) supplemented with 10% heat inactivated fetal bovine serum (FBS, Biowest, Nuaillé, France), 2 mM glutamine (Gibco, Waltham, MA, USA; ID 25030081) and 80 µg/mL gentamicin (Normon Laboratories, Tres Cantos, Spain).

Techniques: Transfection, Immunofluorescence, Flow Cytometry, Viability Assay, Fluorescence, Staining, Microscopy, MTT Assay

Journal: Antioxidants

Article Title: Dipeptide Repeat Pathology in C9orf72 -ALS Is Associated with Redox, Mitochondrial and NRF2 Pathway Imbalance

doi: 10.3390/antiox11101897

Figure Lengend Snippet: DPRs exposure leads to mitochondrial and intracellular superoxide generation in NSC34 cells. ( A ) NSC34 cells were transiently transfected with EGFP (control), EGFP-GR 50 or EGFP-PR 50 during 48 h. or treated with 6 µM of HA, HA-GR 20 or HA-PR 20 peptides for 2 h. ( A , C ) Cells were treated with 1 µM MitoSOX during 30 min and fluorescence was measured by flow cytometry analysis. ( B , D ) cells were treated with 2 µM hydroethidine during the last hour of transfection/treatment and probe intensity was measured by flow cytometry analysis. Each point depicts the geometric mean of probe fluorescence in cells from that sample (( A , B ), alive/EGFP + cells, ( C , D ), all alive cells). Bar height indicates the mean of the whole group and error bars indicate the SEM. Statistical analysis was performed with one-way ANOVA. Dunnett’s post-hoc test. *, p < 0.05; **, p < 0.01; ****, p < 0.0001.

Article Snippet: The Mouse Motor Neuron-Like Hybrid Cell Line (NSC34) (Cedarlane Laboratories, Burlington, Canada) was grown in DMEM (Dulbecco’s modified Eagle’s medium with high glucose; Sigma-Aldrich, San Luis, MO, USA; ID D5648) supplemented with 10% heat inactivated fetal bovine serum (FBS, Biowest, Nuaillé, France), 2 mM glutamine (Gibco, Waltham, MA, USA; ID 25030081) and 80 µg/mL gentamicin (Normon Laboratories, Tres Cantos, Spain).

Techniques: Transfection, Fluorescence, Flow Cytometry

Journal: Antioxidants

Article Title: Dipeptide Repeat Pathology in C9orf72 -ALS Is Associated with Redox, Mitochondrial and NRF2 Pathway Imbalance

doi: 10.3390/antiox11101897

Figure Lengend Snippet: Pharmacological activation of NRF2-dependent transcription by dimethyl fumarate is impaired in EGFP-GR 50 , EGFP-PR 50 or (G 4 C 2 ) 102 expressing cells. ( A ) NSC34 cells were transiently transfected with EGFP, EGFP-GR 50 or EGFP-PR 50 for 48 h and treated with either VEH (DMSO 0.1%) or DMF 100 µM over the last 16 h. ( A ) The levels of the indicated proteins were measured by performing an immunoblot analysis. ACTB levels were determined as a loading control. ( B ) Densitometric quantification for the immunoblots for NRF2 and HO1 presented in ( A ). ( C ) NSC34 cells were transiently co-transfected with EGFP, EGFP-GR 50 and EGFP-PR 50 and plasmids encoding for ARE-luciferase reporter and TK-Renilla luciferase for 48 h and treated as before. Fold of induction over vehicle in relative luciferase units is shown. ( D ) Fold of induction on mRNA levels of the Sqstm1 and Nqo1 genes in EGFP-DPRs transfected cells treated with DMF. mRNA levels were normalized using the geometric mean of the housekeeping genes Gapdh and Actb and to the non-induced control. ( E ) The expression of the sham cassette or the (G 4 C 2 ) 102 cassette was induced for 7 days with 1 µg/mL tetracycline in NSC34 cells. In the last 16 h, cells were treated with either VEH (DMSO 0.1%) or DMF 100 µM. The levels of the indicated proteins were measured by performing an immunoblot analysis. GAPDH levels were determined as a loading control. ( F ) Densitometric quantification for the immunoblots for NRF2 and HO1 presented in ( C ). Individual points in ( B , D ) represent the protein levels normalized by protein amount in each lane, bar height represents the mean of those protein levels in each group and error bars represent the SEM. Statistical analysis was performed with two-way ANOVA. Dunnett’s post-hoc test in ( B – D ). Bonferroni’s post-hoc test in ( F ). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The Mouse Motor Neuron-Like Hybrid Cell Line (NSC34) (Cedarlane Laboratories, Burlington, Canada) was grown in DMEM (Dulbecco’s modified Eagle’s medium with high glucose; Sigma-Aldrich, San Luis, MO, USA; ID D5648) supplemented with 10% heat inactivated fetal bovine serum (FBS, Biowest, Nuaillé, France), 2 mM glutamine (Gibco, Waltham, MA, USA; ID 25030081) and 80 µg/mL gentamicin (Normon Laboratories, Tres Cantos, Spain).

Techniques: Activation Assay, Expressing, Transfection, Western Blot, Luciferase

Journal: Antioxidants

Article Title: Dipeptide Repeat Pathology in C9orf72 -ALS Is Associated with Redox, Mitochondrial and NRF2 Pathway Imbalance

doi: 10.3390/antiox11101897

Figure Lengend Snippet: DPRs do not modify Nfe2l2 mRNA levels, splicing, half-life or sequester them. NSC34 cells were transiently transfected with EGFP (control), EGFP-GR 50 or EGFP-PR 50 for 48h. ( A , B ) Nfe2l2 total transcripts levels and intron levels in mRNA were measured by qRT-PCR. ( C ) Transcription was blocked with 5 µg/mL of actinomycin D in transfected NSC34 cells for the indicated times and mRNA levels for Nfe2l2 were measured by qRT-PCR. ( D ) mRNA levels of the indicated transcripts were measured by qRT-PCR in RNA bound to EGFP-immunocomplexes. In ( A , B ) mRNA levels were normalized by the geometric mean of the housekeeping genes Gapdh , Actb and Tbp. In ( C ) mRNA levels were normalized by the geometric mean of the stable transcripts from Gapdh and Actb . Bar height ( A , B , D ) and point ( C ) indicates the mean of the whole group and error bars indicate the SEM. Statistical analysis was performed with one-way ANOVA for ( A , B ), and two-way ANOVA in ( C ), yielding non-significant changes.

Article Snippet: The Mouse Motor Neuron-Like Hybrid Cell Line (NSC34) (Cedarlane Laboratories, Burlington, Canada) was grown in DMEM (Dulbecco’s modified Eagle’s medium with high glucose; Sigma-Aldrich, San Luis, MO, USA; ID D5648) supplemented with 10% heat inactivated fetal bovine serum (FBS, Biowest, Nuaillé, France), 2 mM glutamine (Gibco, Waltham, MA, USA; ID 25030081) and 80 µg/mL gentamicin (Normon Laboratories, Tres Cantos, Spain).

Techniques: Transfection, Quantitative RT-PCR

Journal: Antioxidants

Article Title: Dipeptide Repeat Pathology in C9orf72 -ALS Is Associated with Redox, Mitochondrial and NRF2 Pathway Imbalance

doi: 10.3390/antiox11101897

Figure Lengend Snippet: General translational shutdown caused by DPRs impairs NRF2 induction by MG132. NSC34 cells were transiently transfected with EGFP, EGFP-GR 50 or EGFP-PR 50 . ( A , B ) transfected NSC34 cells were treated with 1 µg/mL puromycin for 1 h. ( A ) immunocytochemical detection of puromycin-labelled peptides. Yellow arrows, EGFP − cells; blue arrows, EGFP + cells; red arrows, EGFP-GR50 + cells; green arrows, EGFP-PR 50 + cells. Scale-bar: 40 µm; (B) violin plots depicting the puromycin intensity distribution in at least 100 cells in EGFP, EGFP-GR 50 and EGFP-PR 50 conditions. ( C ) transfected cells were treated with MG132 (20 µM) for the indicated times. NRF2 levels were measured by immunoblot. ACTB was measured as a loading control. *, non-specific band. ( D ) densitometric quantification of the immunoblot presented in A. Values are expressed as % NRF2 accumulation at time 4 h. In (B) dotted lines represent the median and the 1st and 3rd quartile. Statistical analysis was performed with one-way ANOVA. Dunnett’s post-hoc test. ****, p < 0.0001. In ( D ) the point represents the mean and error bars represent SEM. Statistical analysis was performed with two-way ANOVA. Dunnett’s post-hoc test. #, EGFP vs. EGFP-GR 50; *, EGFP vs. EGFP-PR 50. * or #, p < 0.05; ##, p < 0.01.

Article Snippet: The Mouse Motor Neuron-Like Hybrid Cell Line (NSC34) (Cedarlane Laboratories, Burlington, Canada) was grown in DMEM (Dulbecco’s modified Eagle’s medium with high glucose; Sigma-Aldrich, San Luis, MO, USA; ID D5648) supplemented with 10% heat inactivated fetal bovine serum (FBS, Biowest, Nuaillé, France), 2 mM glutamine (Gibco, Waltham, MA, USA; ID 25030081) and 80 µg/mL gentamicin (Normon Laboratories, Tres Cantos, Spain).

Techniques: Transfection, Western Blot

Journal: Antioxidants

Article Title: Dipeptide Repeat Pathology in C9orf72 -ALS Is Associated with Redox, Mitochondrial and NRF2 Pathway Imbalance

doi: 10.3390/antiox11101897

Figure Lengend Snippet: Activation of NRF2 rescues from DPRs toxicity. ( A , B ) NSC34 cells were transiently transfected with EGFP, EGFP-GR 50 or EGFP-PR 50 for 48 h or the expression of the Sham cassette or the (G 4 C 2 ) 102 cassette was induced for 7 days with 1 µg/mL tetracycline in NSC34 cells, respectively. Cells were treated with either VEH (DMSO 0.1%) or DMF 100 µM over the last 4 h and stained with 2 µM hydroethidine during the last hour. Probe intensity was measured by flow cytometry analysis. In ( A , B ), each point depicts the geometric mean of probe fluorescence in cells from that sample ( A ), alive EGFP + cells, ( B ), all alive cells). ( C ) NSC34 cells were transduced with either control lentiviral particles or encoding the stable NRF2 version lacking the ETGE KEAP1-binding motif for 48 h and then treated with 6 µM of HA, HA-GR 20 or HA-PR 20 peptides for 24 h. Cell viability was measured using a MTT assay Bar height indicates the mean of the whole group and error bars indicate the SEM. Statistical analysis was performed with one-way ANOVA ( A , B ), Dunnett’s post-hoc test or two-way ANOVA ( C ), Bonferroni’s post-hoc test. ** p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The Mouse Motor Neuron-Like Hybrid Cell Line (NSC34) (Cedarlane Laboratories, Burlington, Canada) was grown in DMEM (Dulbecco’s modified Eagle’s medium with high glucose; Sigma-Aldrich, San Luis, MO, USA; ID D5648) supplemented with 10% heat inactivated fetal bovine serum (FBS, Biowest, Nuaillé, France), 2 mM glutamine (Gibco, Waltham, MA, USA; ID 25030081) and 80 µg/mL gentamicin (Normon Laboratories, Tres Cantos, Spain).

Techniques: Activation Assay, Transfection, Expressing, Staining, Flow Cytometry, Fluorescence, Transduction, Binding Assay, MTT Assay