3 methyladenin

Human prostate carcinoma DU145 (ATCC; HTB-81), PC3 cells (ECCAC; HTB-81) and their drug-resistant sub-lineages were routinely cultivated in DMEM/F12 HAM (Sigma, St. Louis, MO) medium supplemented with 10% fetal bovine serum (FBS) and antibiotics [19 (link),38 (link)]. Human umbilical vein endothelial cells (HUVEC; Life Technologies Corporation, Carlsband, CA, USA) were cultured (up to six passages) in endothelial basal medium (EBM; Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS) and supplement cocktail (hydrocortisone, recombinant hEGF, bovine brain extract, gentamicin, amphotericin-B; all from Lonza [58 (link)]). For endpoint experiments, media supplemented with DCX and/or FF were added to cancer cell cultures at the concentrations given in the text. Culture media were supplemented with: docetaxel (DCX; 0.125–50 nM), mitoxantrone (MTX; 62.5–2000 nM) and/or fenofibrate (FF; 5–25 μM; F6020, Sigma, Saint Louis, MO, USA), GW6471 (10 μM; G5045), N-acetyl-L-cysteine (NAC; 1 mM; A9165, Sigma), elacridar (100 nM); sulfinpyranoze (500 μM), Fumitremorgin C (10 μM), 3-methyladenin (3MA; 0.5 μM; all from Sigma) at the time points indicated in the text. Chemical inhibitors were administrated at the time points indicated in the text and at the concentrations that secure their specific action and the lack of cytotoxic effects.

For immunoblot analysis, anti-SQSTM1, anti-LC3 and anti-BECN1 were purchased from BD Biosciences and Sigma Aldrich, respectively. For immunofluorescence studies, anti-LC3 was purchased from Cell Signaling and anti-SQSTM1 from Abnova. Anti-TLR2 was from Genetex and anti-MyD88 was from Cell Signaling. Monoclonal anti- β-actin was provided by Abcam and a polyclonal anti- GAPDH was from Ambion Life Technologies. Antibodies directed against gD, ICP0, phospho-p38α and histone H3 were from Santa Cruz Biotechnology; the antibody against ICP8 was kindly provided by Pr. Bernard Roizman. Horseradish peroxidase anti-rabbit and anti-mouse antibodies were from Santa Cruz Biotechnology. Tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit antibody was from Jackson and Alexa Fluor 350 donkey anti-mouse antibody was from Invitrogen. Spautin-1 (10 μM), 3-methyladenin (10 mM), filipin (2.5 μg/ml) and sucrose (0.45 M) were from Sigma Aldrich.