mouse neuronal n2a cells  (ATCC)

Journal: Journal of Advanced Research

Article Title: Inhibition of mitophagy via the EIF2S1-ATF4-PRKN pathway contributes to viral encephalitis

doi: 10.1016/j.jare.2024.08.003

Figure Lengend Snippet: HSV-1 neurotropic infection causes mitochondrial damage in vivo and in vitro . ( A ) Distribution of viral gene in different brain region of HSE mice (n = 6). (B) Immunohistochemistry assay was performed to detect mitochondrial TOMM20 in various brain regions, including olfactory bulb (OB), cerebral cortex (CC), and pons/medulla oblongata/cerebellum (P/M/C) of both mock-infected (Ctrl) and HSV-1 infected mice. Scale bar, 100 μm. (C) TEM analysis of mitochondria in the brain tissue from control or HSE mice. ( D ) Quantification of damaged and total mitochondria from 15 fields of view, as well as mitochondrial aspect ratio and form factor. ( E ) Western blot assay of mitophagy-related proteins the brain tissue of control or HSE mice (n = 3). ( F ) RT-qPCR analysis of the mRNA expression levels of PRKN and PINK1 in OB or total brain tissues. ( G ) BV2 and N2a cells were infected with HSV-1 (MOI 0.1, 0.5 and 1) or treated with CCCP (2 μM) for 12 h, followed by staining with JC-1 probe. Flow cytometry was employed to measure the mitochondrial membrane potential. (H) BV2 cells were infected with HSV-1 (MOI=1) for indicated hours and flow cytometry assay was performed to detect the mitochondrial membrane potential. (I) BV2 cells were infected with HSV-1 (MOI=1) for 12 h and RT-qPCR assay was used to detect the relative mRNA expression ratio of the mitochondrial gene mtCO1 and the nuclear gene β-globin . ( J ) Flow cytometry analysis of intracellular ROS production. ( K ) Western blot analysis of proteins involved in mitochondrial fusion or fission. Data are means ± SD (n = 3). * p < 0.05, ** p < 0.01 or *** p < 0.001 versus control group.

Article Snippet: Mouse neuronal N2a cells (CCL-131), Monkey kidney epithelial line Vero (CCL81) and Human microglial HMC3 cells (CRL-3304) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Infection, In Vivo, In Vitro, Immunohistochemistry, Control, Western Blot, Quantitative RT-PCR, Expressing, Staining, Flow Cytometry, Membrane

Journal: Journal of Advanced Research

Article Title: Inhibition of mitophagy via the EIF2S1-ATF4-PRKN pathway contributes to viral encephalitis

doi: 10.1016/j.jare.2024.08.003

Figure Lengend Snippet: HSV-1 inhibits mitophagy. ( A ) BV2 and N2a cells were infected with HSV-1 (MOI=1) for indicated times and the protein expression levels of PINK1, PRKN and TOMM20 were analyzed by Western blot. (B-C) Immunoblot analysis of indicated proteins in BV2 cells infected with HSV-1 for 3 h at various MOIs. ( C ) Immunoblot analysis of indicated proteins in BV2 cells infected with HSV-1 in the presence or absence of CCCP (2 μM) for indicated times. ( D ) BV2 cells were infected with HSV-1 for indicated times and the mRNA expression levels of PINK1 and PRKN were analyzed by RT-qPCR assay. Data are means ± SD (n = 3). * p < 0.05, ** p < 0.01 versus control group. ( E ) TEM analysis of mitochondria in BV2 cells infected with HSV-1 for 24 h. Arrows indicate viral particles. Scale bar, 2 μm. ( F ) Quantification of damaged mitochondria from 10 fields of view, as well as mitochondrial aspect ratio and form factor. * p < 0.05, ** p < 0.01, *** p < 0.001versus control group.

Article Snippet: Mouse neuronal N2a cells (CCL-131), Monkey kidney epithelial line Vero (CCL81) and Human microglial HMC3 cells (CRL-3304) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Infection, Expressing, Western Blot, Quantitative RT-PCR, Control

Journal: Journal of Advanced Research

Article Title: Inhibition of mitophagy via the EIF2S1-ATF4-PRKN pathway contributes to viral encephalitis

doi: 10.1016/j.jare.2024.08.003

Figure Lengend Snippet: Viral proteins ICP34.5 and US11 inhibit ATF4-mediated expression of PINK1/PRKN. ( A-B ) Overexpression of ICP34.5 and US11 inhibit mitophagy. Cells were transfected with ICP34.5 and US11 expression plasmids for 48 h, and Western blot was employed to detect the overexpression efficiency of Flag-tagged ICP34.5 and Flag-tagged US11 proteins (A), and their impact on the EIF2S1-ATF4-PINK1/PRKN axis (B). ( C-D ) RT-qPCR analysis of the mRNA expression levels of PINK1 and PRKN in BV2 and N2a cells transfected with ICP34.5 and US11 expression plasmids for 48 h. ( E-F ) BV2 cells were transfected with negative control (NC) siRNA, ICP34.5 or US11 siRNA for 48 h, and were then infected with HSV-1 (MOI=1) for 24 h. The mRNA expression levels (E) and protein levels (F) of PINK1 and PRKN were assessed. Data are means ± SD (n = 3). * p < 0.05, ** p < 0.01 or *** p < 0.001 versus control group.

Article Snippet: Mouse neuronal N2a cells (CCL-131), Monkey kidney epithelial line Vero (CCL81) and Human microglial HMC3 cells (CRL-3304) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Over Expression, Transfection, Western Blot, Quantitative RT-PCR, Negative Control, Infection, Control

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