NADPH Oxidase

Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) and lucigenin (bis-N-methylacridinium nitrate) are used to detect reactive oxygen species (ROS). Luminol bioluminescence detects mainly to neutrophil myeloperoxidase (MPO) activity, and lucigenin bioluminescence detects NADPH oxidase activity and extracellular superoxide production of macrophages (39 (link),40 (link)). Na-luminol and lucigenin (Santa Cruz Biotechnology) were dissolved in phosphate buffered saline to form 20-mg/ml and 2.5-mg/ml stock solutions, respectively. Anesthetized mice were injected intraperitoneally with 150 mg/kg luminol (days 0, 1, 2, and 4) or 25 mg/kg lucigenin (days 0, 2, 6, and 10). Bioluminescence imaging was performed 10 minutes postinjection using an IVIS Lumina II. Acquisition time was 60 seconds, F-stop 1, Binning 8. Data were analyzed and ROIs were applied; luminescence was expressed as total radiance (photons/second/cm²/steradian) within the ROIs.

Antibodies against the following proteins were purchased from Cell Signaling Technology (Danvers, MA, USA): BAX (1:1000), cleaved-Caspase3 (C-Caspase, 1:1000), total Caspase3 (T-Caspase3, 1:1000), total AKT (T-AKT, 1:1000), phosphorylated AKT (P-AKT, 1:1000), T-mTOR (1:1000), P-mTOR (1:1000), T-P70 (1:1000), P-P70 (1:1000), T-ribosomal protein S6 (T-S6, 1:1000), P-S6 (1:1000), T-4EBP1 (1:1000), P-4EBP1 (1:1000), T-glycogen synthase kinase 3β (T-GSK3β, 1:1000), P-GSK3β (1:1000), 4-Hydroxynonenal (4-HNE, 1:200 for staining), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:1000). Antibodies for FNDC5 (1:1000 for western blot, 1:100 for staining), p67phox (1:1000), superoxide dismutase 1 (SOD1, 1:1000), SOD2 (1:1000), B-cell lymphoma 2 (BCL-2, 1:1000), Nrf2 (1:1000), heme oxygenase-1 (HO-1, 1:1000), Kelch-like ECH-associated protein 1 (Keap1, 1:1000), and heat shock protein 20 (HSP20, 1:1000) were purchased from Abcam (Cambridge, UK). Anti-T-FYN (1:200), anti-P-FYN (1:200), and anti-T-proliferating cell nuclear antigen (PCNA, 1:200) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The secondary antibody used for western blot was purchased from LI-COR Biosciences, whereas anti-rabbit/mouse EnVision^TM+/HRP reagent used for immunohistochemistry was obtained from Gene Technology (Shanghai, China). DOX, irisin, AKT inhibitor (AKT i), rapamycin (Rapa) and dexrazoxane (DEX) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dihydroethidium (DHE) was obtained from Keygen Biotech, and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), malondialdehyde (MDA) assay kit, glutathione (GSH) assay kit, total SOD assay kit and NADPH oxidase assay kit were all purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Phosphoinositide 3-kinase (PI3K) activity ELISA assay kit was obtained from Echelon Biosciences Inc. ApopTag® Plus In Situ Apoptosis Fluorescein Detection Kit was purchased from Millipore (Billerica, MA, USA) and the cell counting kit-8 (CCK-8) was obtained from Dōjindo Laboratories (Kumamoto, Japan).

Primary antibodies against PDI (rabbit monoclonal; 1:1000), Ero1-Lα (rabbit monoclonal; 1:1000), IRE1α (rabbit monoclonal; 1:1000), Calnexin (rabbit monoclonal; 1:600), eIF2α (rabbit polyclonal; 1:800), P-eIF2α (rabbit monoclonal; 1:600), and CHOP (mouse monoclonal; 1:800) were purchased from Cell Signaling Technology, Frankfurt am Main, Germany. Antibodies NADPH 4 oxidase (rabbit polyclonal; 1:800), and Total OXPHOS cocktail antibody (rodent monoclonal; 1:800) were purchased from Abcam, Cambridge, UK. Hsp70 (mouse monoclonal; 1:800), and HO-1 (mouse monoclonal; 1:800) were purchased from Enzo life sciences, Lörrach, Germany. β-actin (rabbit polyclonal; 1:1200) from Novusbio, Centennial, CO, USA, and Vinculin (mouse monoclonal, 1:500) from AbD Serotec, Puchheim, Germany. Where applicable secondary antibodies used during immunoblotting were Donkey anti rabbit (1:8000; Novex), Horse anti mouse (1:2000; Cell Signaling Technology), Donkey anti mouse (1:5000; Bethyl), Goat anti rabbit (1:2000; Cell Signaling Technology), and Donkey anti rabbit (1:4000; Novex).

The receptor nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was obtained from the RCSB Protein Data Bank (https://www.rcsb.org/, accessed on 30 May 2022). Using the Discovery studio, the proteins were first prepared for docking by removing water and ligands, inserting polar hydrogen atoms, and saving them in PDB format. Then, the ligand in SDF format from PubChem (https://pubchem.ncbi.nlm.nih.gov, accessed on 30 May 2022) was downloaded. All the compounds tentatively identified in the GC-MS were docked, and the best docked compounds in terms of best binding affinity were presented in the results. In the preparatory steps of compounds to ligands, these were converted to pdbqt format and then were converted to ligands using the Open Babel. The prepared and optimized ligands were docked blindly in the protein’s grid box to allow them to find any suitable binding location with PyRx [25 ]. The docking studies were validated by superimposing the co-crystallized ligand (Ascorbic acid) with extracted ascorbic acid from the crystal structure and redocking it to the crystal structures of NADPH Oxidase. The Ligplot software was used to visualize 2D structures of ligand–protein interactions [26 (link)].