A003 4 1

BV2 cells were seeded in 6-well plates at a density of 5 × 10⁵ cells/mL and incubated overnight. BV2 cells were infected with different PRV titres for 24 h, or the cells were infected with 1.66 × 10⁶ TCID₅₀ PRV for 6, 12, and 24 h or 1.66 × 10⁶ TCID₅₀ PRV for 24 h, followed by CUR treatment for 6, 12, and 24 h. The supernatant was collected, and NO levels were estimated using an NO detection kit (A003-4–1, A020-2–2, Nanjing Jiancheng Biological Company, China) according to the manufacturer’s protocol.

BV2 and PC-12 cell viability was assessed using CCK-8 reagent (BS350B, Biosharp, China). BV2 cells were seeded at a density of 5 × 10⁴ cells/well in 96-well plates, incubated overnight and treated with varying concentrations of CUR or Compound C for 24 h. Alternatively, PC-12 cells were seeded at a density of 5 × 10⁴ cells/well in 96-well plates, incubated overnight and exposed to the conditioned media (CM) of different BV2 cell phenotypes. CM was obtained from the supernatant of BV2 cells treated with/without PRV and/or CUR. After the PC-12 cells were disrupted and homogenized by a high-speed disperser, LDH activity and MDA levels were determined by a colorimetric method using LDH and MDA detection kits, respectively (A003-4–1, A020-2–2, Nanjing Jiancheng Biological Company, China) according to the manufacturer’s protocol.

PQQ was obtained in the form of a disodium salt from Nascent Health Sciences LCC (NY, USA). Phenylephrine (PE) was from Sigma-Aldrich (MO, USA). Primary antibodies specific for YAP, GPX4, FSP1, and CoQ10 were from ABclonal (A1002, A1933, A12128, and A15193), while antibodies specific for p-YAP, BNP, β-actin, GAPDH, and Histone H3, as well as secondary goat anti-rabbit and goat anti-mouse IgG were from Wuhan Servicebio Technology Co., Ltd. (GB114060, GB11667, GB12001, GB15002, GB11026, GB23303, and GB23301). The 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (S0033) and the mitochondrial membrane potential assay kit JC-1 (C2006) were purchased from the Beyotime Institute of Biotechnology. A test kit for measuring ferrous ion concentration (E-BC-K773-M) was purchased from Elabscience (Wuhan, China), and the kits for measuring malondialdehyde (MDA) and glutathione (GSH) levels were obtained from Nanjing Jiancheng Institute of Biological Engineering (A003-4-1, A006-2–1). Ferrostatin-1(Fer-1) was purchased from Selleck Chemicals (USA). Gibco (CA, United States) was the source of all cell culture reagents.

A 2 mL MAC-T cells suspension (5 × 10⁴ cells/mL) was seeded into the wells of a 6-well plate. After the cell confluence reached 70–80%, the completed medium was removed, and the cells were washed twice with cold PBS. Then, the cells were incubated with treated medium. The MDA content of the MAC-T cells was determined using a microplate method kit (Cat No. A003-4-1; Nanjing Jiancheng Biotechnology Institute, Nanjing, China). The total oxidant status (TOS, Cat No. BPE92369), total antioxidant status (TAS, Cat No. BPE92207), TNF-α (Cat No. BPE92091), IL-1β (Cat No. BPE92157), IL-6 (Cat No. BPE92153), and IL-10 (Cat No. BPE92159) of the MAC-T cells were detected using ELISA kits (Shanghai Lengton Bioscience Co., Ltd., Shanghai, China) following the manufacturer's instructions. Briefly, MAC-T cells were digested by trypsin and placed in centrifuge tubes. Then, the tubes were centrifuged at 2,000 rpm for 20 min, and the supernatant was collected. After the cells were counted, the cell suspension was adjusted with PBS to reach a cell concentration of 1 × 10⁶ cells/mL. Absorbance was detected at 530 (MDA) and 450 nm (TOS, TAS, TNF-α, IL-1β, IL-6, and IL-10) using an enzyme-labeling instrument (BioTek, Hong Kong, China).

BEAS-2B cells were incubated with 10 μmol/L 2′, 7′-Dichlorodihydrofluoresce in diacetate (DCFH-DA) (Beyotime, Nantong, China) for 30 min, avoiding light, and detected by flow cytometry (FACS Calibur, BD FACSCalibur Becton Dickinson, USA) (the excitation wavelength: 488 nm; the emission wavelength: 525 nm) to examine reactive oxygen species (ROS) production. BEAS-2B cell suspension was collected after centrifugation (12000 × g, 10 min). The concentrations of superoxide dismutase (SOD) (A001-3-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and malondialdehyde (MDA) (A003-4-1, Nanjing Jiancheng Bioengineering Institute) and the contents of the nonenzymatic antioxidant system (GSH, GSSG, and GSH/GSSG ratio; Beyotime, Shanghai, China) were detected using commercial assay kits according to the operating instructions.