Pd98059 pd

Anti-MMP-9 polyclonal goat antibody, anti-β-actin monoclonal mouse antibody, goat anti-rabbit IgG/HRP, goat anti-mouse IgG/HRP, rabbit anti-goat IgG/HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Phospho-EGFR rabbit mAb, anti-EGFR rabbit mAb, anti-p44/42 MAP Kinase monoclonal rabbit antibody, anti-Phospho-p44/42 MAP Kinase monoclonal rabbit antibody, anti-AKT monoclonal rabbit antibody, anti-Phospho-AKT monoclonal mouse antibody, anti-Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit mAb, anti-SAPK/JNK rabbit mAb, anti-Phospho-p38 MAPK (Thr180/Tyr182) (12F8) Rabbit mAb and anti-p38 MAPK rabbit antibody were purchased from Cell Signaling Laboratories (Beverly, MA). PMA (Phorbol-12-myristate-13-acetate), specific inhibitors of PI3K [LY294002 (LY)], MAPK family [PD98059 (PD), mitogen-activated protein kinase kinase (MEK) inhibitor; SP600125 (SP), c-jun N-terminal kinase (JNK) inhibitor and SB203580 (SB), P38 MAPK inhibitor], gelatin and FITC-phalloidin were purchased from Sigma Chemical Co, (St.Louis, MO). PC, cholesterol and PEG4000 were purchased from Sigma Chemical Co, (St.Louis, MO); Honokiol was separated and purified by our laboratory and its purity and structure were analyzed and identified by high performance liquid chromatography and nuclear magnetic resonance [16 ].

BC3 (ATCC, CRL-2277) and BCBL1 (kindly provided by Prof. P. Monini, National AIDS Center, Istituto Superiore di Sanità, Rome, Italy) are human B cell lines derived from Primary Effusion Lymphoma (PEL). Fresh PEL cells were obtained from ascites grown in SCID mice. Mice were engrafted with the BC3 cell line; after 30/35, when mice became ill, they were sacrificed, and ascites were withdrawn. Cells were cultured in RPMI 1640 (Sigma, R0883) containing 10% fetal calf serum (Euroclone, ECLS0180L), L-glutamine (2 mM), streptomycin (100 µg/mL), and penicillin (100 U/mL) (Gibco, 10378-016) at 37 °C in 5% CO2. Cells were treated with the following chemicals: Dimethyl fumarate (DMF) (20 and 50 µM; Sigma-Aldrich, St. Louis, MO, USA, cat. n. 242926), Chloroquine (CQ) (20 µM; Sigma-Aldrich, cat. n. C6628), NVP-BEZ235 (BEZ) (dual PI3K/mTOR inhibitor; 100 nM; Selleckchem, Planegg, Germany, cat. n. S1009), Brusatol (BRU) (NRF2 inhibitor; 5 nM; Sigma-Aldrich, St. Louis, MO, USA, cat. n. SML1868), PD98059 (PD) (ERK inhibitor; 20 µM; Sigma-Aldrich, St. Louis, MO, USA, P215).
Cells were treated with DMF for 24 h, Chloroquine was added for the last 18 h, and all inhibitors were added to the cells 30 min before DMF.