Nox2ds tat

Human umbilical vein endothelial cells (HUVECs) (Lonza, Basel, Switzerland, Cat. N. CC-C2517A) were cultured in complete EGM-2 medium (Lonza Cat. No. CC-3162) and 10% FBS and were plated at a density of 2500 cells per cm². HUVECs were seeded and grown overnight to a sub-confluence level.
Before activation, cells were pre-incubated (20 min at 37 °C) with NOX2ds-tat (10 μM; Anaspec, Fremont, CA, USA), a NOX2 inhibitor. After incubation, cells were treated with cortisol (175 or 256 ng/mL) for 3 h.
Conditioned media were collected to quantify soluble biomarkers, such as H₂O₂, sNOX2dp, endothelin-1 and NO, and pellets were analyzed with Western blot in order to verify p-eNOS expression. The experiments were conducted on five different batches of HUVECs.

The Human skeletal muscle myoblasts cell line (HSMM, Lonza, Basel, Switzerland) was cultured in SkBM^TM-2 Basal Medium and SkGM^TM-2 SingleQuots^TM supplements, 2 mM glutamine, and 1% antibiotics (all Gibco) for expansion and maintenance of the undifferentiating state. When cultures reached 80% confluence, myogenic differentiation was induced by replacing the expansion media with DMEM/0.2% FBS. Afterward, cells were pre-treated for 2 h either with vehicle (Phosphate Buffered Saline, PBS) or NOX2ds-tat (10 μM in PBS; Anaspec, Fremont, CA, USA), the most specific inhibitor of interactions between NOX2 and p47phox [28 (link)]. Cells were then stimulated with 10% plasma obtained from 3 different amateurs and 3 different elites’ athletes for 24 h.
Experiments were also conducted by treating cells either with vehicle (ddH₂O) or cocoa-derived polyphenols (1 μM in ddH₂O) for 1 h before stimulation with 10% plasma obtained from 3 amateurs and 3 elites’ athletes for 24 h. The cocoa-derived polyphenols concentration was chosen as the lowest non-toxic concentration to obtain the inhibition. The experiments were conducted on three different batches of HSMM.
Conditioned media were harvested and tested for the quantification of soluble NOX2 and H₂O₂ as described, and pellets were analysed in western blot to verify muscle damage with α-actin expression.