Effect of 4-HNE on cellular cytotoxicity and genotoxicity.

A/ Effect of 4-HNE on cytotoxicity on Co cells. Data are expressed as the mean ± SEM (n = 3). Cell viability assay was done by using cell proliferation reagent WST-1 (Roche Life Science) according to manufacturer’s protocol. Briefly, cells were seeded at the density of 1.3 × 10⁴ cells/ well in 96-well plates and cultured under permissive conditions (33°, IFN-γ) until sub confluence occurred, and then transferred to non-permissive conditions (37°C, without IFN-γ) for 24 h. When cells were confluent, the culture media were replaced with 4-HNE (10–80 μM) in DMEM. Following 24-hour treatments with 4-HNE at 37°C, media were removed and cells were incubated in WST-1 reagent for 1 h in the dark. The absorbance of each well was measured by colorimetric at measurement and reference wavelengths of 440 nm and 690 nm respectively (TECAN, Infinite 200). B-C/Cellular genotoxicity. Genotoxic effects of increasing concentrations of 4-HNE was measured by γH2AX in-cell Western assay according to Khoury et al (S1 Reference). Cells were seeded into 96-well plates at 4×10³ cells per well in permissive conditions. After reached sub confluence, cells were transferred to 37°C and treated with 4-HNE (0, 1, 5 or 10 μM) for 24h. The results are expressed as γH2AX fold induction. (B) Co cells and (C) nF cells. Data are expressed as the mean ± SEM (n = 3).

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