Cell culture and treatment
Mouse embryonic fibroblasts (MEFs) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% glutamine. Cells were seeded in 6-well plates at a density of 2 × 10⁵ cells per well and grown to approximately 80% confluency. Protein Y knockdown was achieved using small interfering RNA (siRNA) at a final concentration of 10 nM, transfected with Lipofectamine 3000 according to the manufacturer’s protocol. Transfection efficiency was validated using fluorescently labeled control siRNA, achieving >90% efficiency, and knockdown efficiency was confirmed via Western blotting, showing an 85% reduction in Protein Y expression compared to controls.
Oxidative Stress Induction
Oxidative stress was induced by treating MEFs with 500 µM hydrogen peroxide (H₂O₂) for 12 hours. Untreated cells served as the control group, while scrambled siRNA-treated cells served as an additional control for knockdown conditions.
Cell Viability Assay
Cell viability was assessed using the MTT assay. After treatment, cells were incubated with 0.5 mg/mL MTT for 3 hours at 37°C, and the resulting formazan crystals were dissolved in dimethyl sulfoxide (DMSO). Absorbance was measured at 570 nm using a microplate reader. Results were normalized to the untreated scrambled siRNA control group to account for baseline viability.
Statistical Analysis
All data were analyzed using GraphPad Prism software. Group comparisons were performed using two-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. For pairwise comparisons, two-tailed t-tests were used. Statistical significance thresholds were defined as follows: *p < 0.05, **p < 0.01, and ***p < 0.001. Results are presented as mean ± SEM from at least three independent biological replicates.
Data Table
https://doi.org/12.5000/zenodo.12345670
