nsc34 neurons  (Cedarlane)

Journal: Bioactive Materials

Article Title: Development of a PTEN -siRNA activated scaffold to promote axonal regrowth following spinal cord injury

doi: 10.1016/j.bioactmat.2026.01.022

Figure Lengend Snippet: PTEN -siRNA nanoparticle transfection enables efficient gene silencing with minimal impact on neuron health (A) PTEN -siRNA nanoparticles (100 nM) demonstrated highly efficient knockdown of target PTEN mRNA in differentiated NSC34 neurons, with peak knockdown on Day 3 for the delivered siRNA [t(2) = −4.840, p = 0.02007; one-tailed] with expression levels returning to baseline by Day 7 [t(2) = 0.05373, p = 0.5190; one-tailed]. RNA fold change and associated error was assessed using the 2 −ΔΔCt method and normalized to GAPDH as per (B) Treatment of neurons with PTEN- siRNA nanoparticles at 100 nM had a non-significant effect on the metabolic activity at 3 [t(2) = 0.6370, p = 0.5893, 95 % CI 74.4820 0.1; • = p ≤ 0.1, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.0001.

Article Snippet: NSC34 neuronal culture and transfections: NSC34 neurons (Cedarlane Cat No. CLU140), a hybrid cell line produced by the fusion of motor neuron enriched embryonic mouse spinal cord cells with mouse neuroblastoma cells, were cultured in growth medium Dulbecco's Modified Eagle Medium (DMEM; Sigma-Aldrich, Ireland) supplemented with 10 % fetal bovine serum (FBS; Labtech UK), 1 % (v/v) L-Glutamine (Sigma-Aldrich, Ireland), and 1 % (v/v) Penicillin- Streptomycin Solution (Sigma-Aldrich, Ireland) in a T-175 cell culture flask (37 °C, 5 % CO 2 ).

Techniques: Transfection, Knockdown, One-tailed Test, Expressing, Activity Assay, Control, Staining

Journal: Bioactive Materials

Article Title: Development of a PTEN -siRNA activated scaffold to promote axonal regrowth following spinal cord injury

doi: 10.1016/j.bioactmat.2026.01.022

Figure Lengend Snippet: Scaffold-mediated siRNA delivery does not affect the viability of neurons, results in prolonged downregulation of PTEN, and stimulates increased expression of BCL2 and GAP43 mRNA . (A) The metabolic activity of NSC34 neurons cultured on PTEN -siRNA activated scaffolds peaked by day 3 post-transfection [t(2) = 4.0599, p = 0.05565, 95 % CI 93.954 0.1; • = p ≤ 0.1, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.0001.

Article Snippet: NSC34 neuronal culture and transfections: NSC34 neurons (Cedarlane Cat No. CLU140), a hybrid cell line produced by the fusion of motor neuron enriched embryonic mouse spinal cord cells with mouse neuroblastoma cells, were cultured in growth medium Dulbecco's Modified Eagle Medium (DMEM; Sigma-Aldrich, Ireland) supplemented with 10 % fetal bovine serum (FBS; Labtech UK), 1 % (v/v) L-Glutamine (Sigma-Aldrich, Ireland), and 1 % (v/v) Penicillin- Streptomycin Solution (Sigma-Aldrich, Ireland) in a T-175 cell culture flask (37 °C, 5 % CO 2 ).

Techniques: Expressing, Activity Assay, Cell Culture, Transfection, Control, Lactate Dehydrogenase Assay, Gene Expression, One-tailed Test

Journal: Brain research bulletin

Article Title: RBM5 induces motor neuron apoptosis in hSOD1 G93A -related amyotrophic lateral sclerosis by inhibiting Rac1/AKT pathways.

doi: 10.1016/j.brainresbull.2024.111049

Figure Lengend Snippet: Fig. 3. Overexpression of RBM5 induces motor neurons apoptosis. (A) Western blotting analysis and qRT-PCR were performed to assess the efficiency of RBM5 overexpression (n=3). (B) Flow cytometry was employed to assess the apoptosis of NSC34 cells following transfection with RBM5 or EV plasmid for 48 h (n=3). (C) TUNEL staining of NSC34 cells after treatment with RBM5 or EV plasmid (n=3). (D) Cell viability was quantified using CCK-8 (n=5). Data are presented as means ± SD. Statistical analysis was performed using student’s t-test. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The NSC34 cell line (Cedarlane Laboratories, Vancouver, Canada) is a hybrid of embryonic mouse neuroblastoma and spinal cord motor neurons.

Techniques: Over Expression, Western Blot, Quantitative RT-PCR, Flow Cytometry, Transfection, Plasmid Preparation, TUNEL Assay, Staining, CCK-8 Assay

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, Phospho-proteomics, Comparison, Staining, Purification, Positive Control, Activity Assay, Control, Immunohistochemical staining, Immunoprecipitation