Journal: Frontiers in Neuroscience
Article Title: A Non-Canonical Role for IRE1α Links ER and Mitochondria as Key Regulators of Astrocyte Dysfunction: Implications in Methamphetamine use and HIV-Associated Neurocognitive Disorders
doi: 10.3389/fnins.2022.906651
Figure Lengend Snippet: IRE1α overexpression augments cytokine expression and increases glutamate clearance in human astrocytes. Backbone and IRE1α transfected astrocytes were treated with IL-1β for 24 h. (A–D) Cells were immunolabeled with antibodies specific for IRE1α (red) and the astrocyte marker glial fibrillary acidic protein (GFAP, green). Nuclear DNA was labeled with DAPI (blue). (E–G) Experiments in four separate biological donors were analyzed to quantify morphological activation. Individual dots on graphs represent compiled fold-changes calculated from duplicate wells and/or triplicate images per condition for each biological astrocyte donor. (E) GFAP intensity was measured across full-well scans using SoftMax Pro and normalized to DAPI. (F) Process length of individual astrocytes was manually traced and measured using ImageJ Software. (G) Percent morphological activation was calculated based on the number of astrocytes presenting with ‘reactive’ morphology divided by the total number of astrocytes imaged. (H) CCL2 and (I) CXCL8 levels were assessed by an ELISA, and expression was normalized to metabolic activity prior to calculating fold changes to backbone. (J) Astrocytes were treated with 400 nM glutamate for 24 h. Remaining glutamate levels were quantified by fluorescent assay to calculate% glutamate clearance followed by fold change for each individual donor. Individual dots on graphs represent compiled data from triplicate experiments per biological astrocyte donor. Significance was determined by one-way ANOVA and Tukey’s post hoc for multiple comparisons.
Article Snippet: Colorimetric enzyme-linked immunosorbent assays (ELISA) were performed according to the manufacturer’s instructions to quantify C-C motif chemokine ligand 2 (CCL2) and C-X-C motif chemokine ligand 8 (CXCL8) secretion levels in culture supernatants (CCL2, cat # DCP00; CXCL8, cat # D8000C, R&D Systems).
Techniques: Over Expression, Expressing, Transfection, Immunolabeling, Marker, Labeling, Activation Assay, Software, Enzyme-linked Immunosorbent Assay, Activity Assay, Fluorescence
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