A061 1

Ascorbate (AsA) and contents of dehydroascorbate (DHA) were measured as reported by Gillespie et al. (2007) (link) with some modifications. Briefly, the roots were homogenized by adding 1.6 mL of 6% trichloroacetic acid (TCA). Afterward, the homogenate was centrifuged at 13,000 rpm for 5 min, 4 ◦C. The absorbance was measured at a wavelength of 525 nm. A standard curve was used to estimate the total ascorbate content of the solution, and DHA concentrations were determined based on deducting the reduced ascorbate value from the total ascorbate value. The contents of glutathione (GSH) and oxidized glutathione (GSSG) were assessed, and detection kits were used for this purpose (A061-1) (Nanjing Jiancheng, China).

All experimental cell lines were suspended in a complete medium, and then equal amounts of cells were allowed for growth in 6-well plates (4 × 10⁵ cells/well) until being completely adherent. Thereafter, they were treated for different time periods (i.e., 0, 4, 16 h) with 50 μM of tBHQ. All the cells were collected in PBS and then subjected to the measurement of total glutathione, reduced glutathione (GSH) and oxidized glutathione (GSSG) by using a glutathione assay kit (A061-1, Nanjing Jiancheng, Nanjing, China) according to the manufacturer’s instruction. Of note, two standards of GSH and GSSG were also prepared in the same assays. The assay was designed by employing an Ellman’s reagent (5,5’-disulfidebis-2-nitrobenzoic acid, DNTB), which can react with GSH to form 2-nitro-5-thiobenzoic acid, a yellow product with an absorbance at a wavelength of 405 nm. In addition, the protein concentrations in all experiment cells were determined by the bicinchoninic acid assay (BCA, P1511, ApplyGene Co., Beijing, China) and used as an internal control for the normalization, along with relevant standard curves, in order to calculate amounts of total glutathione, GSSG, and GSH by the formula provided by this manufacturer. The final resulting data are shown by a ratio of GSSG to GSH levels.