Journal: International Journal of Molecular Medicine

Article Title: The pro-survival pathways of mTOR and protein kinase B target glycogen synthase kinase-3β and nuclear factor-κB to foster endogenous microglial cell protection

doi: 10.3892/ijmm.19.2.263

Figure Lengend Snippet: Figure 3. Inhibition of glycogen synthase kinase-3ß (GSK-3ß) activity enhances microglia integrity during OGD. The GSK-3ß inhibitors SB216763 (SB21, 5 μM) or SB415286 (SB41, 25 μM) were applied to primary microglia 1 h prior to a 6-h period of OGD. Cell survival, DNA fragmentation, and membrane PS exposure were determined 24 h following OGD using the trypan blue dye exclusion method, TUNEL assay, or Annexin-V labeling respectively. (A), Representative images and quantification of data illustrate trypan blue staining in microglia following OGD. Application of SB216763 (SB21, 5 μM) or SB415286 (SB41, 25 μM) significantly decreased cell staining and increased microglial survival during OGD (*P<0.01 vs. control; †P<0.01 vs. OGD). Green arrows indicate trypan blue uptake into microglia while white arrows illustrate absence of trypan blue uptake. (B), Representative images and quantification of data illustrate DNA fragmentation with TUNEL in microglia following OGD. Application of SB216763 (SB21, 5 μM) or SB415286 (SB41, 25 μM) significantly decreased TUNEL staining and decreased microglial DNA fragmentation during OGD (*P<0.01 vs. control; †P<0.01 vs. OGD). Green arrows indicate DNA fragmentation in microglia while white arrows illustrate absence of genomic DNA injury. (C), Representative images and quantification of data illustrate membrane PS exposure with Annexin-V in microglia following OGD. Application of SB216763 (SB21, 5 μM) or SB415286 (SB41, 25 μM) significantly decreased Annexin-V staining and decreased microglial PS externalization during OGD (*P<0.01 vs. control; †P<0.01 vs. OGD). (D), The mTOR inhibitor rapamycin (Rapa) at concentrations of 1.0-20.0 nM in conjunction with SB216763 (SB21, 5 μM) or SH6 (20 μM) was applied to the EOC 2 microglial cell line 1 h prior to a 6-h period of OGD and cell survival was assessed 24 h later. Concurrent inhibition of mTOR activity with rapamycin during SB21 administration reduced the beneficial effects of GSK-3ß inhibition and worsened microglial survival to levels slightly greater than during OGD alone. In contrast, concurrent inhibition of Akt1 activity did not alter protection during GSK-3ß inhibition or lead to a synergistic benefit (*P<0.01 vs. OGD; †P<0.01 vs. OGD/SB21). In all cases, each data point represents the mean and SEM. Control or CON indicates untreated cultures.

Article Snippet: The glycogen synthase kinase (GSK)-3ß inhibitors SB216763 [3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol-3yl)-1H-pyrrole-2,5-dione] (SB21) or SB415286 [3-[(3-Chloro4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole2,5-dione] (SB41) (Tocris, Ellisville, MO) were applied continuously to the microglial cultures 1 h prior to OGD.

Techniques: Inhibition, Activity Assay, Membrane, TUNEL Assay, Labeling, Staining, Control

Journal: The Journal of Biological Chemistry

Article Title: Disruption of lysosomal nutrient sensing scaffold contributes to pathogenesis of a fatal neurodegenerative lysosomal storage disease

doi: 10.1016/j.jbc.2024.105641

Figure Lengend Snippet: Reagent resources and list of primers

Article Snippet: Mouse monoclonal anti-GSK-3ß (3D10) , Cell signaling , Cat#9832; RRID: AB_.

Techniques: Electron Microscopy, Recombinant, Protease Inhibitor, Staining, Isolation, Clinical Proteomics, Membrane, Enzyme-linked Immunosorbent Assay, Activation Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Disruption of lysosomal nutrient sensing scaffold contributes to pathogenesis of a fatal neurodegenerative lysosomal storage disease

doi: 10.1016/j.jbc.2024.105641

Figure Lengend Snippet: Reagent resources and list of primers

Article Snippet: Rabbit monoclonal anti-GSK-3ß (D5C5Z) , Cell signaling , Cat#12456; RRID: AB_2636978.

Techniques: Electron Microscopy, Recombinant, Protease Inhibitor, Staining, Isolation, Clinical Proteomics, Membrane, Enzyme-linked Immunosorbent Assay, Activation Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Disruption of lysosomal nutrient sensing scaffold contributes to pathogenesis of a fatal neurodegenerative lysosomal storage disease

doi: 10.1016/j.jbc.2024.105641

Figure Lengend Snippet: Reagent resources and list of primers

Article Snippet: Rabbit monoclonal anti-phospho-GSK-3ß (Ser9) (D85E12) , Cell signaling , Cat#5558; RRID: AB_.

Techniques: Electron Microscopy, Recombinant, Protease Inhibitor, Staining, Isolation, Clinical Proteomics, Membrane, Enzyme-linked Immunosorbent Assay, Activation Assay, Mutagenesis