Ag 20b 0153

The role of Trolox and Irisin in preventing and/or counteracting cell damage induced by RPM exposure was studied by treating cell cultures with Trolox, r-Irisin or both. Specifically, cells were seeded in a 24-well plate at a density of 4 × 10⁴ cells/well. Cell cultures were incubated with 1 × 10⁻⁴ M Trolox (S-238815, Sigma Aldrich, St. Louis, MO, USA), 10 ng/mL r-Irisin (AG-20B-0153, AdipoGen® Life Sciences, Liestal, Switzerland) or combined treatment, and exposed to RPM for 48 h. Brightfield images of differentiated HT22 cells before and after exposure to RPM were acquired at 40x magnification using NIS-Elements software (5.30.01; Laboratory Imaging, Prague, Czech Republic) and shown in S1 Fig for each experimental condition. Subsequently, treated cell cultures were subjected to the same experimental procedures as untreated samples.

The role of irisin in preventing damage induced by RPM exposure was investigated by treating primary cultures of human osteoblasts with r-irisin. Specifically, cells from the first or second passage were seeded in a 24-well plate at a density of 2 × 10⁴ cells/well. Primary cultures derived from each patient group were incubated with 10 ng/mL r-irisin (AG-20B-0153, AdipoGen^® Life Sciences, Liestal, Switzerland) and exposed to RPM for 3 days or 6 days. Subsequently, the r-irisin-treated cell cultures were subjected to the same experimental procedures as the untreated samples.