Plumbagin

NOX4 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Novus Biologicals (Littleton, CO, USA) and Abcam (Cambridge, UK). NOX2 antibody was purchased from Abcam. Phosphorylated EGFR antibodies (Y845, Y1068, and Y992), Hoechest 33342 and Alexa Fluor 488 goat anti-mouse IgG were purchased from Invitrogen. Anti-EGFR, anti-c-Src, anti-p22^phox, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, HRP-conjugated goat anti-rabbit IgG antibodies were obtained from Santa Cruz Biotechnology. Anti-Bim antibody was purchased from Enzo Life Sciences (Farmingdale, NY). anti-Akt, Anti-phospho Akt (S473), anti- ERK1&2, anti-phospho ERK1&2 (T202/Y204), anti-phospho Src (Y416), anti-phospho EGFR (Y1068), anti-phospho GSK3β (S9), anti-STAT3, and anti-cleaved caspase-3 were obtained from Cell Signaling Technology (Beverly, MA). Anti-EGFR (528) antibody was purchased from Abcam. Anti-β-actin antibody, Dimethyl sulfoxide (DMSO), N-acetylcysteine (NAC), Diphenyleneiodonium (DPI), apocynin, plumbagin and Poly (2-hydroxyethyl methacrylate) (Poly-HEMA) were purchased from Sigma (St. Louis, Missouri).

Gallic acid, ellagic acid, piperine, β-asarone (Merck, Darmstadt, Germany); plumbagin (Sigma-Aldrich, Seelze, Germany); acetonitrile and phosphoric acid (Labscan, Bangkok, Thailand); purified water was prepared by Milli Q^® system from Millipore (Bedford, MA, USA); murine macrophage leukemia cell line (RAW 264.7: ATCC^® TIB-71™; American Type Culture Collection, VA, USA); Roswell Park Memorial Institute medium 1640 (RPMI-1640), fetal bovine serum (FBS), penicillin-streptomycin (P/S), and 0.5% trypsin-EDTA (Gibco BRL Life Technologies, NY, USA); phosphate buffer saline (PBS; Amresco, Ohio, USA); dimethyl sulfoxide (DMSO; Fluka, Munich, Germany); LPS and 3-(4,5-dimethyl-2-thiazolyl)-2,5 diphenyl-2H-tetrazolium bromide (MTT) (Sigma-Aldrich Inc., MO, USA); PGE₂ EIA kit (monoclonal; Cayman Chemical Company, MI, USA); mouse TNF-α Quantikine ELISA Kit (R&D Systems Inc., MN, USA).

Plumbagin was purchased from Sigma–Aldrich (St. Louis, MO, USA). Primary antibodies against MMP-2, MMP-9, cleaved caspase-3, Bcl-2, Bax, β-actin, Bad, Bcl-xL were procured from Abcam, USA. TNF-α, NF-κBp65, IκBα, p-IκBα, p-IKKα, IKKα p-IKKβ, IKKβ, and horseradish peroxidase-labeled IgG secondary antibodies were procured from Cell Signaling Technology Inc. (Danvers, MA, USA). ELISA kits for determination of cytokines -TNF-α, IL-1β and IL-6 were obtained from Biolegend (San Diego, CA, USA). Cell lysis buffer for Western blotting analysis was purchased from Beyoime Institute of Biotechnology (Beijing, China). All the other chemicals used for analysis were from Sigma–Aldrich, if otherwise are specified.

A quantity of 1g dried samples subjected to cold extraction using 100 ml chloroform for 72 h. The supernatant was collected by decantation into an amber colored bottle. The crude extracts were filtered by filter paper (Whatman, no. 1), evaporated by the rotary evaporator, dissolved separately in 5 ml methanol and filtered through 0.45 µm membranes before injection to the HPLC. A Breeze HPLC system from Waters Corporation (USA) was used at a wavelength of 270 nm with the outputs to record and analyze using a software (Breeze, USA). Plumbagin as standard was purchased from Sigma-Aldrich. An amount of 50 µL of methanolic extract of sample was injected into C8 analytical column (250 mm * 4.6 mm, particle size 5 µm; Perfectsill, MZ-Analysentechnik, Germany). The mobile phase was a mixture of methanol: water (80:20) and run at the isocratic condition with a flow rate of 0.9 ml min^-1. The chromatographic peak of Plumbagin was confirmed by comparing its retention time with that of the standard (31 ). Quantitative estimation of Plumbagin was carried out based on the peak area of specific concentrations of the sample and the standard. The area under the peaks of Plumbagin was integrated and converted to concentration using its calibration curve.