Experimental MethodsLipotoxicity assayThe CHO cells were seeded at a density 3 × 105 cells per 35-mm dish and incubated at 37 °C in a CO2incubator. After 24 h, cells were treated with various concentrations of VLCFAs prepared as FA/BSA complexes using the FA/IP/BSA method 2–4) for the indicated durations. In a separate set of experiments, C18:1 or C22:1 was co-incubated with VLCFAs for the same periods. Control cells were treated with vehicle (BSA + IP) only. Following incubation, cell viability was assessed using the trypan blue dye exclusion assay, and results were expressed as percentages relative to vehicle-treated controls.Determination of FA incorporation into cellular lipidsCHO cells were seeded at 1 × 106 cells per 100-mm dish containing 10 mL of FBS-supplemented medium and incubated for 24 h at 37 °C in a CO2 incubator. Cells were then serum-starved for 24 h, after which VLCFAs were added as FA/BSA complexes prepared via the FA/IP/BSA method 2–4). In subsequent experiments, C18:1 or C22:1 was co-incubated with VLCFAs at the indicated concentrations. After incubation, cellular lipids were extracted, subjected to methanolysis and analyzed by GC. Lipidomics analysisThe lipids extracted from cultured cells were obtained using the Bligh and Dyer method 6). These extracted lipids were then analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The obtained lipidomics data were processed using metabolomics software, MS-DIAL.Electron microscopy and qPCRCHO-zp102 cells were treated with C24:0, with or without C18:1 in serum-free medium. Total RNA was extracted (QIAGEN), and cDNA was synthesized (TOYOBO). qPCR was performed using StepOnePlus (Thermo Fisher), with 18S rRNA as the internal control. For electron microscopy, cells were similarly treated, then fixed, postfixed, stained, dehydrated, and embedded in resin. Ultrathin sections were imaged using a JEM-1400 TEM (JEOL) following established protocols 7).Results and DiscussionVLCFA toxicity and reduction in cellular C18:1Our results revealed that peroxisome-deficient cells are more sensitive to VLCFA-induced toxicity than wild-type cells, highlighting the role of peroxisomal β-oxidation in mitigating VLCFA cytotoxicity. Significant reductions in cell viability were observed when peroxisome-deficient cells were incubated with ≥20 μM C22:0, 100 μM C24:0 and 150 μM C26:0 (Fig. 1A, B). In these experiments, remarkable decreases in the level of cellular C18:1 FA were observed. Such decreases were also observed in the experiments with toxic concentrations of monounsaturated VLCFAs (C22:1, C24:1 and C26:1 FA) in peroxisome-deficient cells (Fig. 1A, B). These results suggest a potential link between C18:1 depletion and VLCFA-induced cytotoxicity.― 266 ―
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