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. 2022 Nov 26;59:102547. doi: 10.1016/j.redox.2022.102547

Fig. 6.

Fig. 6

FABP5 is involved in mitochondrial damage and lipid peroxidation in SH-SY5Y cells. (A) Representative Western blots of FABPs and β-actin in mouse brains and SH-SY5Y cells. (B) Quantitative analyses of FABP5 protein expression in cells transfected with FABP5 shRNA (n = 4). (C) SH-SY5Y cell viability was assessed using CCK-8 assays. Tested cells were pcDNA 3.1-transfected cells (mock, HY08, and NAC) and FABP5 shRNA-transfected cells (shFABP5) (n = 8) (D and E) Representative images of JC-1 green and red fluorescence in SH-SY5Y cells (D). Scale bar = 20 μm. Quantification of the ratio of the red to green JC-1 fluorescence intensity (E, n = 300 cells). Data are presented as mean ± SEM of 300 cells counted per group. (F) SH-SY5Y cell viability after transfection with pcDNA 3.1 (mock) or FABP5 overexpression plasmid (FABP5) (n = 8) (G and H) Representative images of green and red JC-1 fluorescence (G) and quantification of the ratio of JC-1 fluorescence intensities in FABP5-overexpressing SH-SY5Y cells (H, n = 300 cells). Data are presented as mean ± SEM of 300 cells counted per group. (I–K) Representative immunofluorescence staining of 4-HNE (green), Mito-tracker (red), and FABP5 (blue) in rotenone-treated SH-SY5Y cells (I). Scale bar = 30 μm. 4-HNE and Mito-tracker co-localization (J) and 4-HNE fluorescence intensity (K) were quantified using Image J software (n = 4). *p < 0.05, **p < 0.01 vs. vehicle-treated mock group; ##p < 0.01 vs. rotenone-treated mock group. (L) Quantification of intracellular 4-HNE levels in fatty acid (AA, DHA, LA or AO)-treated SH-SY5Y cells (n = 3). *p < 0.05, **p < 0.01 vs. control cells. AA: arachidonic acid, DHA: docosahexaenoic acid, LA: linoleic acid, OA: oleic acid. (M) Quantification of mitochondrial 4-HNE levels in AA (50 μM)-treated SH-SY5Y cells in the presence or absence of HY08 (or FABP5 shRNA) (n = 3). **p < 0.01 vs. vehicle-treated mock group; #p < 0.05, ##p < 0.01 vs. AA-treated mock group. The data shown in each column represent the mean ± SEM. (N) Schematic diagram of the involvement of FABP5 in reducing membrane potential and lipid peroxidation during oxidative stress. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)