L name

MDA-MB-231, MCF-7 and HCC38 cells were plated at a density of 5 × 10⁴ per well and incubated for 24 h. In order to understand the time course during which ROS/RNS generation occurred, cells were exposed to cortisol (1 μM) and NE (1 μM) for 15, 30, and 90 minutes. Control wells were left untreated. Following this period, the medium was removed and cells lysed using 500 μl lysis buffer (Trevigen). Lysates were then collected and ROS/RNS levels were quantified using multiple-step amperometry using a stainless steel counter electrode and non-leak Ag|AgCl reference electrode. Measurements of the current were obtained at +0.3 V, +0.45 V, +0.62 V and +0.85 V for a duration of 30 s.
Additional measurements were also carried out in MDA-MB-231 and MCF-7 cells to understand how ROS/RNS levels were altered when the cells were incubated with RU486 (1 μM) (GR antagonist), propranolol (1 μM) (β-adrenergic receptor antagonist) 1400 W dihydrochloride (10 μM) (iNOS inhibitor; Tocris, UK), L-NAME (100 μM) (non-specific NOS inhibitor; Tocris, UK) and PP2 (10 μM) (Src inhibitor; Abcam, UK) for 30 minutes prior to hormone treatment.

Anti-GAPDH antibody was purchased from Fitzgerald Antibodies (Acton, MA) and was used at a dilution of 1:1,000. Anti-phospho-Akt (Ser473), anti-Akt, and anti-phospho-eNOS (Ser1177) antibodies were purchased from Cell Signaling (Danvers, MA) and were all used at a dilution of 1:1,000. Anti-eNOS antibody (1:500 dilution), anti-mouse IgG (1:10,000 dilution), and anti-rabbit IgG (1:2,000 dilution) antibodies were purchased from Abcam (Cambridge, MA). MK2206 and GDC0068 were purchased from SelleckChem (Houston, TX). Gallein, LY294002, L-NAME, and ODQ were purchased from Tocris (Ellisville, MO). All other chemicals were purchased from Sigma (St. Louis, MO).