P iκb

Urolithin A (≥97% purity by HPLC, Figure 1A) was purchased from Sigma-Aldrich (St. Louis, MO, USA), cell viability assays (MTT), and poly(I:C) were purchased from InvivoGen (San Diego, CA, USA). Enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (Minneapolis, MN, USA). DAPI solution was purchased from Sigma-Aldrich (St. Louis, MO, USA). The inhibitor PD98059 was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Antibodies against β-actin, TLR3, TRIF, IRF, STAT1, NF-κB, I-κB, COX-2, iNOS, Nrf2, and phosphorylated IRF (pIRF), STAT1, NF-κB (pNF-κB), and IκB (pIκB) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against JNK, ERK, p38, and phosphorylated JNK (pJNK), ERK (pERK), and p38 (pp38) were purchased from Millipore (Billerica, MA, USA).

Alpha-phenyl-N-t-butylnitrone (PBN) and ethanol were from Sigma Chemical Co. (St. Louis, MO). Antibodies against Akt, pAkt and CYP2E1 were from Cell Signaling Technology (Beverley, MA). Antibodies against HO-1, SREBP-1, NF-κB p65, p-IκB, MyD88, β-actin and Lamin A/C were from Santa Cruz Biotechnologies (Santa Cruz, CA). TRI reagent was from Molecular Research Center, Inc (Cincinnati, Ohio). RNase-free DNase was from Promega Corporation (Madison, WI). Chemiluminescence detection kit was from Pierce Biotechnology (Rockford, IL). Oil Red O was from Sigma Chemical Co. (St. Louis, MO). All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO) if not otherwise stated.

U87MG (a human primary glioblastoma cell line) was obtained from the Bioresource Collection and Research Center (Food Industry Research and Development Institute, Hsinchu, Taiwan). HBVP (a human brain vascular pericyte cell line), HA (a human astrocyte cell line), and HBMEC (a human brain microvascular endothelial cell line) were purchased from ScienCell Research Laboratories Inc. (Carlsbad, CA, USA). Gibco™ Eagle's minimum essential medium (MEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Pericyte medium (PM), astrocyte medium (AM), and endothelial cell medium (ECM) were obtained from ScienCell Research Laboratories Inc. (Carlsbad, CA, USA). Primary antibodies against I_κB kinase (IKK), I_κB, p65, p-IKK, p-I_κB, p-p65, lamin B, and β-actin were obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). The Bradford protein assay reagent for protein concentration determination was purchased from Bio-Rad Laboratories Inc. (Hercules, CA, USA). PG, propidium iodide (PI), TMZ, 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA), dimethyl sulfoxide (DMSO), Trypan blue solution, crystal violet, and other chemicals were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA).

Heart issues or RAW 264.7 macrophage cells were lysed with a Radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors. For nuclear and cytoplasmic protein analyses, the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Jiangsu, China) was used. Total protein concentrations were determined with the BCA protein assay kit (Beyotime). Protein samples (50 μg) were subjected to electrophoresis by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes, and blocked in Tris-buffered saline containing 0.05% tween and 5% non-fat dry milk. Primary antibody (Gal-3, Abcam, 1:5,000; p-IκB, Santa Cruz, 1:500; IκB, Proteintech, 1:1,000; p-NF-κB p65, AFFINITY, 1:500; p50, Proteintech, 1:200; b-actin Atagenix, 1:3,000; GAPDH, Proteintech, 1:5,000) incubations were performed at 4°C overnight, and secondary antibodies were incubated for 1 h at room temperature. The immunoreactive bands were visualized using Enhanced Luminol Reagent and Oxidizing Reagent. Densitometric analyses were conducted using Quantity One software (Bio-Rad, Hercules, CA, USA).

Western blot analysis was done as described previously [8 (link)]. The membrane was incubated with specific antibodies: mouse monoclonal antibodies against MMP9, p-p65, p65, p-IκB, STAT3, p-STAT3, histone H1 and β-actin (1:500 dilution, Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA), Goat rabbit polyclonal MMP3 (1:500 dilution, Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA), rabbit polyclonal for p50, p-IKKα, IKKα, p-IKKβ, IKKβ, p-IκB and IκB (1:500 dilution, Santa Cruz Biotechnology Inc.) and iNOS and COX-2 (1:1000 dilution, Cayman Chemical, Ann Arbor, Mich, USA). The relative density of the protein bands was scanned by densitometry using MyImage (SLB, Seoul, Korea) and quantified by Labworks 4.0 software (UVP, Inc., Upland, CA, USA).