A015 3 1

Cultured SWFs were collected and homogenized with PBS. Subsequently, centrifugation was performed at 800 rpm at 4 °C for 10 min, and the supernatant was aspirated to obtain a 10% tissue homogenate. The tissue homogenate was used to measure oxidation parameters and determine total protein concentration. The activity of CAT (A00171-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and T-SOD (A001-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), full antioxidant capacity (T-AOC) (A015-3-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), the concentrations of MDA (A003-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), GSH (A006-2-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and hydrogen peroxide (H₂O₂) (A064-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and total protein concentration were determined using kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. Additionally, the cultured SWFs were homogenized in boiling water, heated in boiling water for 10 min, and then subjected to centrifugation. The ATP level was determined by an ATP assay kit (A095-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), following the instructions provided by the manufacturer.

GCs incubated with 0, 4, 20, 100 and 500 ng/mL PRL were cultured in a 6-well plate at a density of 6 × 10⁴ per well and then trypsinized and collected in 1.5 ml centrifuge tubes. After incubation, cells were immediately centrifuged twice at 1000 × g for 10 min to remove the supernatant to obtain GCs, which were kept at −80 °C prior to analysis.
Superoxide dismutase (SOD) activity (A001-3; Nanjing Jiancheng Bioengineering Institute, Jiangsu, China), malondialdehyde (MDA, Nanjing Jiancheng Bioengineering Institute, A003-1) and total antioxidant capacity (T-AOC) (A015-3-1, Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) were measured following the manufacturer’s instructions with respective analytical kits.