Brusatol

Reagent kits to detect D-lactate acid (D-LA), intestinal fatty acid binding protein (I-FABP), diamine oxidase (DAO), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione/L-glutathione (oxidized) (GSH/GSSG) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). A nuclear extract kit was purchased from Beyotime Institute of Biotechnology (Haimen, China). Antibodies to microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, p62, p-Akt (Ser⁴⁷³), Akt, p-GSK-3β (Ser⁹), GSK-3β, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Nrf2, heme oxygenase 1 (HO-1), NAD(P)H: quinine oxidoreductase 1 (NQO1), and LaminB1 were purchased from Cell Signaling Technology (Danvers, MA, USA). A LI-COR IRDye800CW goat anti-rabbit secondary antibody was obtained from LI-COR Biosciences (Lincoln, NE, USA). CQ was purchased from Cell Signaling Technology, Danvers, MA, USA. The autophagy inducer rapamycin and inhibitor 3-methyladenine (3-MA) were from Selleck Chemicals (Houston, TX, USA). SC66 and brusatol, specific antagonists of Akt and Nrf2, respectively, were purchased from Sigma-Aldrich, Merck KGaA (Darmstadt, Germany). All chemicals used were of the highest grade available.

The human NSCLC cells cell lines HCC827, H460, and A549 were purchased from American Type Culture Collection (ATCC, USA). These cells were cultured in RPMI1640 (Welgene, Daegu, South Korea) supplemented with 10% foetal bovine serum (FBS) (Welgene, Daegu, South Korea) and 1% penicillin/streptomycin at 37 ℃ in a humidified atmosphere containing 5% CO₂. Brusatol (Carbosynth, UK) was purchased and dissolved in dimethyl sulfoxide (DMSO) (D2650, Sigma Aldrich, USA). MG132 was purchased from CalBiochem (SanDiego, CA, USA). Nrf2-EGFP and Keap1-Flag vectors were purchased from Addgene (http://www.addgene.org). The oligo ribonucleotide sequences of human Nrf2 siRNA (siNrf2) were as follows: 5′-UCUGACUCCGGCAUUUCACUTT-3′ (sense) and 5′-AGUGAAAUGCCGGAGUCAGATT-3′ (antisense) (Shanghai GenePharma Co. Ltd., China).

Human B cell lines derived from KSHV-positive PEL cell lines, BC3, and BCBL-1 (kindly supplied by Prof. P. Monini, National AIDS center, Istituto Superiore di Sanità, Rome, Italy) were grown in RPMI 1640 medium (Sigma-Aldrich, Burlington, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, Burlington, MA, USA), L-glutamine (2 mM) (Aurogene, Rome, Italy), and streptomycin/penicillin (100 μg/mL) (Aurogene, Rome, Italy) (complete medium) at 37 °C in a 5% CO₂ humified setting. Cells were seeded into 6-well plates at a density of 4 × 10⁵ per well in a final volume of 2 mL in complete medium and were treated for 24 h (h) singly or in combinations with the STAT3 inhibitor tyrphostin AG490 (50–100–200 µM) (Calbiochem, San Diego, CA, USA; 658411) and the NFE2L2 inhibitor brusatol (10–20–40 nM) (Sigma Aldrich, St. Louis, MO, USA; 1868). In some experiments, to evaluate the role of HSP90A and HSPB1 in the mechanism/s to which NFE2L2 could sustain STAT3 activity and vice versa, PEL cells were treated for 24 h with inhibitors of HSPB1 and HSP90A, respectively, J2 (10 µM) (MedChemExpress, Monmouth Junction, NJ, USA; HY-124653), 17-AAG (0.1 µM) (Selleckem, Planegg, Germany; S1141). All the drugs were dissolved in DMSO, and the control cells were supplemented with DMSO in the same amount used for the other samples.

Antibodies used were: anti-Nox4 (Abcam Cat# ab133303, RRID:AB_11155321), anti-Nrf2 (Abcam Cat# ab62352, RRID:AB_944418), anti-Keap1 (Proteintech Cat# 10,503–2-AP, RRID:AB_2132625), anti-beta-actin (Cell Signaling Technology Cat# 3700, RRID:AB_2242334), anti-p62 (Cell Signaling Technology Cat# 5114, RRID:AB_10624872), and Phospho-p62 (Ser349) (Cell Signaling Technology Cat# 16,177, RRID:AB_2798758). Secondary horseradish peroxidase conjugated antibody used were anti-rabbit (1:5000, Thermo Fisher Scientific, Cat# G-21234, RRID AB_2536530) and anti-mouse (1:6000, Thermo Fisher Scientific Cat# A24524, RRID:AB_2535993).
Brusatol was purchased from Sigma Aldrich (SML1868) and was prepared in DMSO (5 mg/ml). DMF was purchased from Sigma-Aldrich (242,926) and was dissolved in DMSO (5 mg/ml). The selective Nox4 inhibitor GKT137831 was kindly supplied by Genkyotex (S.A., Geneva, Switzerland).

Sulforaphane (SFN) (Cat #S4441, Sigma-Aldrich, St. Louis, MO, USA) and Brusatol (BRU) (Cat #SML1868, Sigma-Aldrich) were solubilized in DMSO and used to treat cells based on our prior findings [12 (link)]. All treatments were prepared in traditional cell culture growth media composed of high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) with sodium pyruvate (Cat #25-500, Genesee Scientific, San Diego, CA, USA) and 10% fetal bovine serum (Cat #F4135, Genesee Scientific). Before treatment, MACT cells were seeded in 6-well plates at a seeding density of 3 × 10⁵ cells per well in DMEM media and grown overnight in an incubator at 37℃/5%CO₂. Three replicates of MACT cells were then treated with either 10 µM SFN, 100 nM BRU, or an equivalent amount of DMSO (i.e., control) for 24 h before the isolation of RNA. Cells were cultured, and treatments were applied in 2 mL of media/well.

Anti-β-actin antibody (A1978) was purchased from Sigma-Aldrich (St. Louis, MO, USA), and anti-nuclear factor erythroid 2-related factor 2 (NRF2) antibody (#12721), anti-glucocorticoid receptor (GR) antibody (#12401), anti-phospho-c-Jun antibody (#9261), anti-c-Jun antibody (#9165), and anti-survivin antibody (#2808) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Dexamethasone (Fuji Pharma Co., Ltd., Tokyo, Japan) was dissolved in phosphate-buffered saline (PBS) to prepare a 1 mM stock solution. 5-FU, gemcitabine, 2′,7′-dichlorofluorescein diacetate (DCF-DA), N-acetyl-cysteine (NAC), and brusatol were purchased from Sigma-Aldrich, and SP600125 was purchased from Merck Millipore (Darmstadt, Germany). They were dissolved in dimethyl sulfoxide (DMSO) to prepare 200 mM 5-FU, 1 mM gemcitabine, 20 mM DCF-DA, 5 M NAC, and 50 mM brusatol stock solutions.

LPS (E. coli: serotype O55:B5), apomorphine, DAPI, PLD (>95% purity), and Brusatol (BT, an Nrf2 inhibitor) were obtained from Sigma-Aldrich (St. Louis, MO, USA). MK2206 (an AKT inhibitor) and NP-12 (Tideglusib, a GSK-3β inhibitor) were purchased from Selleck, 0.25% trypsin were obtained from Invitrogen (Carlsbad, CA, USA). Penicillin-streptomycin (PS) solutions and phosphate buffered saline (PBS) were provided by (Beyotime Inst. Biotech, Beijing, China). The Fetal bovine serum (FBS) and Dulbecco's Modified Eagle's Medium (DMEM) were provided by Gibco (Grand Island, NY, USA). Rat or mouse PGE2, IL-6, IL-1β, and TNF-α ELISA kits were obtained from Biolegend (San Diego, CA, USA).