Journal: Journal of Functional Foods

Article Title: Krill oil attenuates diabetes-associated cognitive dysfunction by inhibiting microglial polarization-induced neuron injury

doi: 10.1016/j.jff.2024.106064

Figure Lengend Snippet: Fig. 5. KO had no protective effect on AGEs-activated neuron apoptosis. (A) Diagram for culture and treatment of N2a cells; (B) TUNEL staining with (C) percentage of positive cells quantified; (D) protein levels of BAX, BCL2 and cleaved Caspase3. The data were summarized as means ± SD (n = 3). *P < 0.05 vs. Ctrl; #P < 0.05 vs. AGEs + Veh. Abbreviations: AGEs, advanced glycation end products; Veh, vehicle (glycerol). Other abbreviations are the same as in Fig. 4.

Article Snippet: Mouse neuron N2a cells were obtained from The American Type Culture Collection (ATCC, CCL131, Manassas, Virginia, USA).

Techniques: TUNEL Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: Multiple sequences orchestrate subcellular trafficking of neuronal PAS domain–containing protein 4 (NPAS4)

doi: 10.1074/jbc.RA118.001812

Figure Lengend Snippet: Subcellular distribution of full-length NPAS4. Expression of YFP-tagged full-length NPAS4 revealed distinct localization of fluorescence. Subcellular localization of expressed proteins in COS-7 and N2a cells was analyzed by confocal microscopy 20–24 h after transfection. Nuclei and nucleoli were stained by Draq5. Bar , 10 μm. A , representative images (single confocal plane) of the subcellular distribution of YFP-NPAS4 in COS-7 cells ( a ) and N2a cells ( b ) in low-glucose medium. B , representative images (single confocal plane) of the subcellular distribution of YFP-NPAS4 in cells cultured in high-glucose medium. Distribution of YFP-NPAS4 in COS-7 cells is shown under normal conditions ( a ), after LMB addition ( b ), and after methanol addition ( c ). C , distribution YFP-NPAS4 in N2a cells under normal conditions ( a and a ′), after LMB addition ( b ), and after methanol addition ( c ). D , representative images (single confocal plane) of the subcellular distribution of YFP-NPAS4 in cells cultured in high-glucose medium. Distribution in COS-7 cells is depicted under normal conditions ( a ), after LMB addition ( b ), and after methanol addition ( c ). Distribution in N2a cells is depicted under normal conditions ( d ), after LMB addition ( e ), and after methanol addition ( f ).

Article Snippet: African green monkey kidney fibroblasts COS-7 (ATCC CRL-1651) and mouse albino neuroblastoma N2a cells (ECACC, Sigma-Aldrich) were cultured in DMEM with 1.0 or 4.5 g/liter glucose and l -glutamine (Lonza).

Techniques: Expressing, Fluorescence, Confocal Microscopy, Transfection, Staining, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Multiple sequences orchestrate subcellular trafficking of neuronal PAS domain–containing protein 4 (NPAS4)

doi: 10.1074/jbc.RA118.001812

Figure Lengend Snippet: Hierarchy of NLS and NES signals in NPAS4. Subcellular localizations of the expressed YFP-tagged derivatives of NPAS4 were analyzed by confocal microscopy 20–24 h after transfecting COS-7 and N2a cells. A , schematic presentation of NPAS4 and YFP-tagged NPAS4 derivatives. B , subcellular distributions of YFP-tagged derivatives of NPAS4. Representative images (single confocal plane) of typical (presented by more than 95% of cells unless stated otherwise) subcellular distributions of the NPAS4 derivatives are presented. Nuclei and nucleoli were stained by Draq5. Bar , 10 μm. Distributions in COS-7 cells in low-glucose medium: YFP-NPAS4/1–144 ( a ), YFP-NPAS4/60–144 ( b ), YFP-NPAS4/145–276 ( c ), YFP-NPAS4/195–340 ( d ), YFP-NPAS4/145–340 ( e ), YFP-NPAS4/1–340 ( f ), YFP-NPAS4/1–580 ( g ), YFP-NPAS4/341–802 ( h ), and YFP-NPAS4/60–802 ( i ). Distributions in N2a cells in high-glucose medium: YFP-NPAS4/1–144 ( a ′), YFP-NPAS4/60–144 ( b ′), YFP-NPAS4/145–276 ( c ′), YFP-NPAS4/195–340 ( d ′), YFP-NPAS4/145–340 ( e ′), YFP-NPAS4/1–340 ( f ′), YFP-NPAS4/1–580 ( g ′), YFP-NPAS4/341–802 ( h ′), and YFP-NPAS4/60–802 ( i ′).

Article Snippet: African green monkey kidney fibroblasts COS-7 (ATCC CRL-1651) and mouse albino neuroblastoma N2a cells (ECACC, Sigma-Aldrich) were cultured in DMEM with 1.0 or 4.5 g/liter glucose and l -glutamine (Lonza).

Techniques: Confocal Microscopy, Staining

Journal: PLoS ONE

Article Title: The Influence of Pathological Mutations and Proline Substitutions in TDP-43 Glycine-Rich Peptides on Its Amyloid Properties and Cellular Toxicity

doi: 10.1371/journal.pone.0103644

Figure Lengend Snippet: (A) The comparison of cell viability in the presence of D1, G294V, G295S, GGG294PPP, and GGG308PPP for 72 hours determined by AlamarBlue assay. Results were means ± SEM of three independent experiments (* = p<0.05, ** = p<0.01, N.S. = not significant). (B) Time-lapse DIC microscopy of N2a cell after the addition of PBS, GGG308PPP, or G295S for 36 hours.

Article Snippet: Mouse neuroblastoma (N2a) cells (generous gift from Dr. Yijuang Chern, IBMS, Academia Sinica) were cultured in Dulbecco's modified Eagle's medium (HyClone) supplemented with 4 mg/L L-glutamine, 1% Penicillin/Streptomycin (Invitrogen) and 10% fetal bovine serum (Invitrogen).

Techniques: Comparison, Alamar Blue Assay, Microscopy

Journal: Nature Communications

Article Title: Localized molecular chaperone synthesis maintains neuronal dendrite proteostasis

doi: 10.1038/s41467-024-55055-7

Figure Lengend Snippet: a Schematic of the pulldown strategy used to identify RBPs binding to the Hspa8 3′ UTR, and a table of the RBPs identified as specifically bound to the Hspa8 3′ UTR in extracts from Ctrl (black *) and MG132-stressed (red *) N2A cells by MS (Created in BioRender. ALAGAR, L. (2024) https://BioRender.com/f90a722 ). Proteomics data is provided in Source data. b Pulldown experiments to validate the binding of STAU2 and FUS to the Hspa8 3′ UTR were analyzed by western blot in two independent replicates. I input, PD pulldown. c , d Representative images of primary mouse motor neurons expressing GFP (Ctrl) or GFP and shRNAs against STAU2 ( c ) or FUS ( d ). Three days after microinjection, stress was induced with 10 μM MG132 for 7 h, and Hspa8 mRNA expression was detected by smFISH. Scale bars = 5 μm. e – h Quantification of the densities of H s pa8 ( e , g ) and eEf1a1 ( f , h ) mRNAs per pixel of soma or dendrite area in Ctrl and MG132-stressed motor neurons expressing GFP with and without the indicated shRNA expression plasmids. For STAU2, the data are the mean ± SEM of three independent experiments (neurons GFP control n = 33, GFP MG132 n = 27, STAU2 shRNA control n = 18, STAU2 shRNA MG132 n = 23; dendrites GFP control n = 105, GFP MG132 n = 111, STAU2 shRNA control n = 72, STAU2 shRNA MG132 n = 98; dots indicate individual soma and dendrite values). P values for Hspa8 mRNA (**** P < 0.0001, ns no significant ( P = 0.99)) and for eEf1a1 mRNA (Soma: ** P < 0.01 (0.031), ns no significant ( P = 0.26), Dendrites: * P < 0.05 (0.076), ns no significant ( P = 0.98). For FUS, five independent experiments (neurons GFP control n = 69, GFP MG132 n = 72, FUS shRNA control n = 75, FUS shRNA MG132 n = 69; dendrites GFP control n = 246, GFP MG132 n = 232, FUS shRNA control n = 255, FUS shRNA MG132 n = 224). P values for Hspa8 mRNA (Soma: ns no significant ( P = 0.88 in Ctrl and 0.1 in MG132), Dendrites: *** P < 0.001; ns no significant ( P = 0.905)) and for eEf1a1 mRNA (Soma: ns no significant ( P = 0.99 in Ctrl and MG132), Dendrites: ns no significant ( P = 0.99 in Ctrl and 0.34 in MG132)) (by 1-way ANOVA). i Ratio of the dendrite to soma Hspa8 mRNA density calculated by averaging the number of mRNAs/pixel of all dendrites of a neuron and dividing it by the number of mRNA/pixel of soma. The data are the mean ± SEM of three independent experiments (GFP control n = 70, GFP MG132 n = 72, FUS shRNA control n = 75, FUS shRNA MG132 n = 69 neurons) from G. * P < 0.05 ( P = 0.021); ns no significant ( P = 0.13) (by 1 way ANOVA). j Representative dendrites from Ctrl and MG132-stressed motor neurons expressing the proteostasis reporter plasmid FLUC-GFP with and without FUS knockdown. GFP aggregation is proportional to proteostasis loss. Scale bar = 10 μm. k Quantification of the GFP signal granularity (the coefficient of variation) in each dendrite in I. The data are the mean ± SEM of four independent experiments (GFP control n = 97, GFP MG132 n = 138, FUS shRNA control n = 88, FUS shRNA MG132 n = 115 dendrites). **** P < 0.0001;** P < 0.01 ( P = 0.0021) (by 1 way ANOVA). Source data are provided as a Source Data file.

Article Snippet: HSPA8 3 ′ UTR PP7-HSPA8 and PP7-LacZ RNA were first PCR amplified from N2A genomic DNA or a plasmid (donated by Dr. Jerry Pelletier) using the primers listed below, and then in vitro transcribed with a MEGAshortscriptTM T7 Transcription Kit (Invitrogen, #AM1354) following the manufacturer’s instructions.

Techniques: Binding Assay, Western Blot, Expressing, Microinjection, shRNA, Control, Plasmid Preparation, Knockdown