Journal: Regenerative Therapy

Article Title: Tanshinone IIA protects motor neuron-like NSC-34 cells against lipopolysaccharide-induced cell injury by the regulation of the lncRNA TCTN2/miR-125a-5p/DUSP1 axis

doi: 10.1016/j.reth.2023.03.007

Figure Lengend Snippet: LPS induces cell injury and inhibits TCTN2 expression in NSC-34 cells. NSC-34 cells were treated with LPS for 24 h at various concentrations of 0, 25, 50, 75 and 100 ng/mL. (A) Cell viability by CCK-8 assay. (B) Representative images depicting a cell apoptosis assay and cell apoptosis by flow cytometry. (C) The levels of TNF-α, IL-6 and IL-1β by ELISA assay using the assay kits. (D) qRT-PCR for relative TCTN2 expression in treated cells. LPS: lipopolysaccharide. ∗ P < 0.05.

Article Snippet: The mouse motor neuron-like NSC-34 cell line was provided by Cedarlane (Burlington, NC, USA) and cultivated at 37 °C, 5% CO 2 in Dulbecco's modified Eagle Medium (Life Technologies, Scotland, UK) plus 1% penicillin/streptomycin (Life Technologies) and 10% fetal calf serum (Biosera, Boussens, France).

Techniques: Expressing, CCK-8 Assay, Apoptosis Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Regenerative Therapy

Article Title: Tanshinone IIA protects motor neuron-like NSC-34 cells against lipopolysaccharide-induced cell injury by the regulation of the lncRNA TCTN2/miR-125a-5p/DUSP1 axis

doi: 10.1016/j.reth.2023.03.007

Figure Lengend Snippet: TSIIA alleviates cell damage induced by LPS in NSC-34 cells. NSC-34 cells were simultaneously treated with 100 ng/mL of LPS and the indicated doses (0, 2, 4, 6 and 8 μM) of TSIIA for 24 h. (A) CCK-8 assay for the viability of cells after various treatments. (B) Representative images showing a cell apoptosis assay and cell apoptosis by flow cytometry. (C) ELISA assay for TNF-α, IL-6 and IL-1β levels using the corresponding assay kit. (D) Relative expression of TCTN2 by qRT-PCR in treated NSC-34 cells. LPS: lipopolysaccharide, TSIIA: Tanshinone IIA.∗ P < 0.05.

Article Snippet: The mouse motor neuron-like NSC-34 cell line was provided by Cedarlane (Burlington, NC, USA) and cultivated at 37 °C, 5% CO 2 in Dulbecco's modified Eagle Medium (Life Technologies, Scotland, UK) plus 1% penicillin/streptomycin (Life Technologies) and 10% fetal calf serum (Biosera, Boussens, France).

Techniques: CCK-8 Assay, Apoptosis Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

Journal: Regenerative Therapy

Article Title: Tanshinone IIA protects motor neuron-like NSC-34 cells against lipopolysaccharide-induced cell injury by the regulation of the lncRNA TCTN2/miR-125a-5p/DUSP1 axis

doi: 10.1016/j.reth.2023.03.007

Figure Lengend Snippet: TSIIA alleviates LPS-induced cell injury by elevating TCTN2 expression. NSC-34 cells were transfected with or without si-NC or si-TCTN2 and then treated with or without LPS (100 ng/mL) and TSIIA (0 or 8 μM). (A) TCTN2 expression by qRT-PCR in transfected cells. (B) Cell viability by CCK-8 assay. (C) Representative images showing a cell apoptosis assay and cell apoptosis by flow cytometry. (D) ELISA assay for TNF-α, IL-6 and IL-1β levels using the corresponding assay kit. (E) Representative images depicting a western blot assay and the levels of PCNA and Bcl-2 by western blot. (F) SOD activity using the assay kit. (G) MDA level using the assay kit. LPS: lipopolysaccharide, TSIIA: Tanshinone IIA. ∗ P < 0.05.

Article Snippet: The mouse motor neuron-like NSC-34 cell line was provided by Cedarlane (Burlington, NC, USA) and cultivated at 37 °C, 5% CO 2 in Dulbecco's modified Eagle Medium (Life Technologies, Scotland, UK) plus 1% penicillin/streptomycin (Life Technologies) and 10% fetal calf serum (Biosera, Boussens, France).

Techniques: Expressing, Transfection, Quantitative RT-PCR, CCK-8 Assay, Apoptosis Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot, Activity Assay

Journal: Regenerative Therapy

Article Title: Tanshinone IIA protects motor neuron-like NSC-34 cells against lipopolysaccharide-induced cell injury by the regulation of the lncRNA TCTN2/miR-125a-5p/DUSP1 axis

doi: 10.1016/j.reth.2023.03.007

Figure Lengend Snippet: TCTN2 directly targets miR-125a-5p. (A) Sequence of miR-125a-5p, the binding sites for miR-125a-5p in TCTN2 and the mutation of the target sites. (B) qRT-PCR for miR-125a-5p relative expression in NSC-34 cells transfected with miRNA NC mimic or miR-125a-5p mimic. (C) Dual-luciferase reporter assays in NSC-34 cells with TCTN2 luciferase reporter construct (WT-TCTN2) or mutant TCTN2 reporter construct (MUT-TCTN2). (D) Relative expression of miR-125a-5p by qRT-PCR in NSC-34 cells exposed to 100 ng of LPS or control for 24 h. (E) qRT-PCR for miR-125a-5p expression in NSC-34 cells treated with LPS or LPS + TSIIA (8 μM). (F) TCTN2 expression by qRT-PCR in NSC-34 cells transfected with pc-NC or pc-TCTN2 (TCTN2 overexpression plasmid). (G) The level of miR-125a-5p by qRT-PCR in NSC-34 cells transfected with pc-NC, pc-TCTN2 (TCTN2 overexpression plasmid), pc-TCTN2+miRNA NC mimic or pc-TCTN2+miR-125a-5p mimic before LPS exposure. LPS: lipopolysaccharide, TSIIA: Tanshinone IIA. ∗ P < 0.05.

Article Snippet: The mouse motor neuron-like NSC-34 cell line was provided by Cedarlane (Burlington, NC, USA) and cultivated at 37 °C, 5% CO 2 in Dulbecco's modified Eagle Medium (Life Technologies, Scotland, UK) plus 1% penicillin/streptomycin (Life Technologies) and 10% fetal calf serum (Biosera, Boussens, France).

Techniques: Sequencing, Binding Assay, Mutagenesis, Quantitative RT-PCR, Expressing, Transfection, Luciferase, Construct, Over Expression, Plasmid Preparation

Journal: Regenerative Therapy

Article Title: Tanshinone IIA protects motor neuron-like NSC-34 cells against lipopolysaccharide-induced cell injury by the regulation of the lncRNA TCTN2/miR-125a-5p/DUSP1 axis

doi: 10.1016/j.reth.2023.03.007

Figure Lengend Snippet: Enforced expression of TCTN2 alleviates LPS-induced cell injury through miR-125a-5p. NSC-34 cells were transfected with pc-NC, pc-TCTN2 (TCTN2 overexpression plasmid), pc-TCTN2+miRNA NC mimic or pc-TCTN2+miR-125a-5p mimic and then exposed to 100 ng/mL of LPS for 24 h. (A) CCK-8 assay for cell viability. (B) Representative images showing a cell apoptosis assay and cell apoptosis by flow cytometry. (C) ELISA assay for TNF-α, IL-6 and IL-1β levels using the corresponding assay kit. (D) Representative images depicting a western blot assay and the levels of PCNA and Bcl-2 by western blot. (E) SOD activity using the assay kit. (F) MDA level using the assay kit. LPS: lipopolysaccharide. ∗ P < 0.05.

Article Snippet: The mouse motor neuron-like NSC-34 cell line was provided by Cedarlane (Burlington, NC, USA) and cultivated at 37 °C, 5% CO 2 in Dulbecco's modified Eagle Medium (Life Technologies, Scotland, UK) plus 1% penicillin/streptomycin (Life Technologies) and 10% fetal calf serum (Biosera, Boussens, France).

Techniques: Expressing, Transfection, Over Expression, Plasmid Preparation, CCK-8 Assay, Apoptosis Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot, Activity Assay

Journal: Regenerative Therapy

Article Title: Tanshinone IIA protects motor neuron-like NSC-34 cells against lipopolysaccharide-induced cell injury by the regulation of the lncRNA TCTN2/miR-125a-5p/DUSP1 axis

doi: 10.1016/j.reth.2023.03.007

Figure Lengend Snippet: TCTN2 targets miR-125a-5p to regulate the expression of DUSP1. (A) Sequence of miR-125a-5p, the binding sites for miR-125a-5p in DUSP1 3′UTR and the mutation of the target sites. (B) Dual-luciferase reporter assays in NSC-34 cells with DUSP1 3′UTR luciferase reporter construct (WT-DUSP1 3′UTR) or mutant DUSP1 3′UTR reporter construct (MUT-DUSP1 3′UTR). (C) Relative expression of DUSP1 protein by western blot in NSC-34 cells exposed to 100 ng/mL of LPS or control for 24 h. (D) Western blot for DUSP1 protein level in NSC-34 cells treated with LPS or LPS + TSIIA (8 μM). (E) DUSP1 protein level by western blot in NSC-34 cells transfected with si-NC or si-DUSP1. (F) qRT-PCR for miR-125a-5p expression in NSC-34 cells transfected with inhibitor NC or miR-125a-5p inhibitor. (G) Western blot for DUSP1 protein level in LPS-exposed NSC-34 cells transfected with inhibitor NC, miR-125a-5p inhibitor, miR-125a-5p inhibitor + si-NC or miR-125a-5p inhibitor + si-DUSP1. (H) DUSP1 protein level by western blot in LPS-treated NSC-34 cells transfected with pc-NC, pc-TCTN2 (TCTN2 overexpression plasmid), pc-TCTN2+miRNA NC mimic or pc-TCTN2+miR-125a-5p mimic. LPS: lipopolysaccharide, TSIIA: Tanshinone IIA. ∗ P < 0.05.

Article Snippet: The mouse motor neuron-like NSC-34 cell line was provided by Cedarlane (Burlington, NC, USA) and cultivated at 37 °C, 5% CO 2 in Dulbecco's modified Eagle Medium (Life Technologies, Scotland, UK) plus 1% penicillin/streptomycin (Life Technologies) and 10% fetal calf serum (Biosera, Boussens, France).

Techniques: Expressing, Sequencing, Binding Assay, Mutagenesis, Luciferase, Construct, Western Blot, Transfection, Quantitative RT-PCR, Over Expression, Plasmid Preparation

Journal: Regenerative Therapy

Article Title: Tanshinone IIA protects motor neuron-like NSC-34 cells against lipopolysaccharide-induced cell injury by the regulation of the lncRNA TCTN2/miR-125a-5p/DUSP1 axis

doi: 10.1016/j.reth.2023.03.007

Figure Lengend Snippet: The reduced expression of miR-125a-5p alleviates LPS-induced cell damage by up-regulating DUSP1. NSC-34 cells were transfected with inhibitor NC, miR-125a-5p inhibitor, miR-125a-5p inhibitor + si-NC or miR-125a-5p inhibitor + si-DUSP1 and then exposed to 100 ng/mL of LPS for 24 h. (A) Cell viability by CCK-8 assay. (B) Representative images showing a cell apoptosis assay and cell apoptosis by flow cytometry. (C) ELISA assay for TNF-α, IL-6 and IL-1β levels using the corresponding assay kit. (D) Representative images depicting a western blot assay and the levels of PCNA and Bcl-2 by western blot. (E) SOD activity using the assay kit. (F) MDA level using the assay kit. LPS: lipopolysaccharide. ∗ P < 0.05.

Article Snippet: The mouse motor neuron-like NSC-34 cell line was provided by Cedarlane (Burlington, NC, USA) and cultivated at 37 °C, 5% CO 2 in Dulbecco's modified Eagle Medium (Life Technologies, Scotland, UK) plus 1% penicillin/streptomycin (Life Technologies) and 10% fetal calf serum (Biosera, Boussens, France).

Techniques: Expressing, Transfection, CCK-8 Assay, Apoptosis Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot, Activity Assay

Journal: Regenerative Therapy

Article Title: Tanshinone IIA protects motor neuron-like NSC-34 cells against lipopolysaccharide-induced cell injury by the regulation of the lncRNA TCTN2/miR-125a-5p/DUSP1 axis

doi: 10.1016/j.reth.2023.03.007

Figure Lengend Snippet: Tanshinone IIA protects LPS-induced NSC-34 cell injury by regulating the lncRNA TCTN2/miR-125a-5p/DUSP1 axis.

Article Snippet: The mouse motor neuron-like NSC-34 cell line was provided by Cedarlane (Burlington, NC, USA) and cultivated at 37 °C, 5% CO 2 in Dulbecco's modified Eagle Medium (Life Technologies, Scotland, UK) plus 1% penicillin/streptomycin (Life Technologies) and 10% fetal calf serum (Biosera, Boussens, France).

Techniques:

Journal: Antioxidants

Article Title: Post-Translational Modification Analysis of VDAC1 in ALS-SOD1 Model Cells Reveals Specific Asparagine and Glutamine Deamidation

doi: 10.3390/antiox9121218

Figure Lengend Snippet: Sequence coverage map of VDAC1 from NSC34, NSC34-SOD1WT and NSC34-SOD1G93A cell lines obtained by tryptic and chymotryptic digestion. The solid line indicates sequence that was obtained from tryptic peptides; dotted lines: sequence obtained from chymotryptic peptides. Unique tryptic (indicated in bold) peptides originated by missed-cleavages were used to distinguish and cover sequences shared by isoforms. Sequences shared by more isoforms are reported in red. The numbering of the sequence considered the starting methionine residue, which is deleted during protein maturation.

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

Techniques: Sequencing, Residue

Journal: Antioxidants

Article Title: Post-Translational Modification Analysis of VDAC1 in ALS-SOD1 Model Cells Reveals Specific Asparagine and Glutamine Deamidation

doi: 10.3390/antiox9121218

Figure Lengend Snippet: Ratio of the absolute intensities of the molecular ions of the sulfur-containing tryptic peptides found in the analysis of VDAC1 from NSC34-SOD1G93A cells HTP-preparation reduced with DTT, carboxyamidomethylated and digested in-solution.

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

Techniques: Sequencing

Journal: Antioxidants

Article Title: Post-Translational Modification Analysis of VDAC1 in ALS-SOD1 Model Cells Reveals Specific Asparagine and Glutamine Deamidation

doi: 10.3390/antiox9121218

Figure Lengend Snippet: MS/MS spectrum of the triply charged molecular ion at m/z 730.6730 (calculated 730.6734) of the VDAC1 tryptic peptide from NSC34-SOD1G93Acellcontaining Asn residue 37 in the deamidated form. 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 asterisk.

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

Techniques: Tandem Mass Spectroscopy, Residue

Journal: Antioxidants

Article Title: Post-Translational Modification Analysis of VDAC1 in ALS-SOD1 Model Cells Reveals Specific Asparagine and Glutamine Deamidation

doi: 10.3390/antiox9121218

Figure Lengend Snippet: Ratio of the absolute intensities of the molecular ions of tryptic peptides containing deamidated against not deamidated amino acids, found in the analysis of VDAC1 from NSC34-SOD1G93A cell HTP-preparation reduced with DTT, carboxyamidomethylated and digested in-solution.

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

Techniques: Sequencing

Journal: Antioxidants

Article Title: Post-Translational Modification Analysis of VDAC1 in ALS-SOD1 Model Cells Reveals Specific Asparagine and Glutamine Deamidation

doi: 10.3390/antiox9121218

Figure Lengend Snippet: Asparagine (N) and glutamine (Q) deamidation in the VDAC1 from NSC34 cells HTP-preparations. Mean, standard deviation, (95%) lower and upper confidence level and the number of scansions are reported.

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

Techniques: Standard Deviation, Residue

Journal: Antioxidants

Article Title: Post-Translational Modification Analysis of VDAC1 in ALS-SOD1 Model Cells Reveals Specific Asparagine and Glutamine Deamidation

doi: 10.3390/antiox9121218

Figure Lengend Snippet: Relative percentage of each VDAC isoform in the three cell lines.

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

Techniques:

Journal: Antioxidants

Article Title: Post-Translational Modification Analysis of VDAC1 in ALS-SOD1 Model Cells Reveals Specific Asparagine and Glutamine Deamidation

doi: 10.3390/antiox9121218

Figure Lengend Snippet: Modified residues found in VDAC1 from NSC34, NSC34-SOD1WT and NSC34-SOD1G93A cell lines. The ratio between modified and unmodified residues is shown in brackets; where the ratio is not indicated the residue is only present in a single form. The dashes indicate residues found exclusively in non-modified form. The asterisk indicates a fully carboxyamidomethylated form.

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

Techniques: Modification, Residue