Journal: Heliyon
Article Title: Adult aberrant astrocytes submitted to late passage cultivation lost differentiation markers and decreased their pro-inflammatory profile
doi: 10.1016/j.heliyon.2024.e30360
Figure Lengend Snippet: Functional parameters and pharmacological response upon long lasting cultivation. ( A ) Increased proliferation rate in HP vs. LP cultures assessed with BrdU immunolabeling and determined as the percentage of BrdU positive cells related to total DAPI positive cells (150 cells per replica, 3 replicas per condition, 3 independent experiments). Values represented were related to proliferation rate of LP cells (100 %). ( B ) Significantly augmented ER stress in HP vs. LP cells evaluated by GRP78/BiP immunostaining (150 cells per replica, 3 replicas per condition, 3 independent experiments). Data found in LP cells were assigned as 100 %. ( C ) Preserved mitochondrial functionality in HP vs. LP cultures assessed by MTT assay. Results obtained indicate absence of changes when comparing LP vs. HP averaged values (5 replicas per passage, 7 independent experiments). ( D ) Effects of 100 μmol/L Zinc chloride (Zn), 10 μmol/L Forskolin (FSK), 2.5 μmol/L Tunicamycin (TN) and 300 μmol/L 6-aminonicotinamide (6AN) on LP and HP cells′ survival assessed with the sulforhodamine B (SRB) method. Except for Zn, SRB values increased in HP vs. LP cultures indicating more living instead of deadly cells (5 replicas per condition and passage, 7 independent experiments). ( E ) Selective vulnerability of AbAs to violacein in comparison to primary astrocytes from adult NoTg animals (AA). Results obtained indicate that LP and HP AbAs′ showed differences in viability at 150 nmol/L violacein; and that the viability of LP, but not of HP AbAs, decreased upon the exposure to 150 and 300 nmol/L violacein (5 replicates of AA, LP and HP per condition, 7 independent experiments). The 100 % value in the y-axis (dashed line) correspond to the viability obtained in AA in control conditions. Values represented in min to max box plots were obtained after pooling the data of each experiment (taken as a single value) and calculating the averaged value of all independent experiments. Data from LP cells were represented in pale green colors whereas those of HP cells are in more intense green, and those from AA appear in blue. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Article Snippet: Then cells were incubated with one or two of the following antibodies: 1:500 anti-GLT1 (Cell Signaling, #3838); 1:600 anti-S100β (Sigma, S2532); 1:500 anti-GS (abcam, 49873); 1:300 anti-MMP-2 (Thermo Fisher Scientific, 436000), 1:500 anti-MMP-9 (Thermo Fisher Scientific, PA5-13199) or 1:300 anti-78-kDa glucose-regulated protein (GRP78/BiP, abcam, ab-21685).
Techniques: Functional Assay, Immunolabeling, Immunostaining, MTT Assay, Comparison
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