T buooh

Dose-dependent oxidative experiments were performed by exposing plasma samples from a healthy subject to increasing concentration of tert-Butyl hydroperoxide (t-BuOOH). Briefly, t-BuOOH (Sigma–Aldrich, USA) was added to plasma samples to reach the final concentration of 0.1, 0.5 or 1 mM. Samples not treated with t-BuOOH were used to determine basal HA isoforms relative abundance. Each mixture was then incubated at 37 °C for 24 hours and, afterwards, analyzed by means of LC-MS according to the method described above.

293T HEK, MEF and derivative cell lines were cultured in Dulbecco's minimal essential medium (Gibco Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) and 2 mM l-glutamine. MEF cells were established from day 14.5 embryos and spontaneously immortalized by serial passaging.
For viability assays, 293T HEK or MEF cells were exposed to MMS, MNNG, H₂O₂ or t-buOOH (all chemicals from Sigma, St. Louis, MO, USA) in serum-free medium for 1 h followed by replacement with medium containing serum. UV treatment was performed using a UV cross-linker (Stratagene, San Diego, CA, USA) set at a 254-nm wavelength. HEK 293T and MEF cells were analyzed 24-h post-treatment using either a Muse flow cytometer with the Muse Count and Viability Assay (EMD Millipore, Billerica, MA, USA) or a Coulter counter coupled with Trypan blue staining. For time-lapse video microscopy, cells were plated onto a 96-well plate for 24 h before treatment with 1.0 mM MMS and video capture at 20-min intervals on a Cellomics Array Scan (ThermoFisher Scientific, Waltham, MA, USA).

Terrific broth, Isopropyl β-D-thiogalactoside, sodium phosphate, hydrogen peroxide, thioflavin-T, t-BuOOH and CumOOH were obtained from Sigma Chemical (St. Louis, MO, USA).