Methods Materials RPMI 1640 medium, Dulbeccos modified Eagles medium, fetal bovine serum, and streptomycin penicillin for cell culture were obtained from PAA Labo ratories. Wang resin was obtained from Synthesis Technologies Inc. DMAP, DCC and DMF were purchased from Dikma Technologies selleck chemicals Tofacitinib Inc. Fmoc L Gly OH and HBTU came from Tianmapharma Co, Ltd. Piperidine and NMM were obtained from Sinopharm Chemical Reagent Co, Ltd. Carboxyfluorescein diacetate, succinimidyle ester was from Molecular Probe and 7 Amino actinomycin D was purchased from Anaspec. Novobiocin and dimethyl sulfoxide were purchased from Sigma. 17 allylamino 17 demethoxygel danamycin was obtained from Invivogen. Anti HSF1 antibody was obtained from Cell Signaling Technology Inc.
Anti HSP70, anti HSP40, anti HSP90, and anti p HSF1 antibodies were purchased from ENZO Life Sciences Inc. Anti p HSF1 was pur chased from Abcam. Nuclear and Cytoplasmic Protein Extraction Kit, Inhibitors,Modulators,Libraries BCA protein assay reagent kit and Beyo ECL Plus for western blot were pur chased from Beyotime Biotechnology. Phosphatase inhibitor cocktail tablets were obtained from Roche. All reagents were stored ac cording to manufacturer recommendations. Celastrol was extracted as previously reported by us. Celastrol and 17 AAG were dissolved in 50 mM and 1 mg ml in DMSO, respectively. NB was dissolved in ddH2O. All of these drugs were stored at 20 C and used within 3 months of preparation. The stored solu tion was further diluted with RPMI 1640 medium or DMEM to a proper lower concentration immediately be fore experiments.
Cell culture and treatment The seven kinds of human cancer cell lines used in this study were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences, including breast cancer cell lines MCF 7 and MDA MB 468, prostate cancer cell line PC3, Inhibitors,Modulators,Libraries hepatic cancer cell line HepG2, leukemic cell lines THP 1, U937, and NB4. Cells were maintained in RPMI 1640 or DMEM supplemented with 10% FBS, 100 IU ml penicillin and 100 ug ml streptomycin in a humidified 5% CO2 incubator at 37 C. Exponentially growing cells were used for experiments. Cells were seeded into 96 well or 6 well culture plates followed by exposure to the indicated doses of celastrol, 17 AAG, or NB for the indicated times. The culture medium with DMSO served as control. The final concentration of DMSO never exceeded 0. 1%.
Each experiment was repeated at least three times. Western blot Cells were incubated in lysis Inhibitors,Modulators,Libraries buffer and cleared by centri fugation Inhibitors,Modulators,Libraries at 13,000 g for 10 min. For the phosphorylation protein assay, phosphatase inhibitor was added to sup press the activity of phosphatase. The extraction of cyto plasmic Inhibitors,Modulators,Libraries and nuclear protein was performed according to product manufacturer instructions. A BCA protein assay though reagent kit determined protein concentrations. Aliquots of samples were subjected to 10% SDS polyacrylamide gels and then transferred to polyvinylidenedifluoride membranes.