Proteasome inhibitor mg132

The snap-frozen myocardial tissue samples were homogenized in a relaxing buffer (90 mM HEPES, 126 mM KCl, 36 mM NaCl, 1 mM MgCl, 50 mM EGTA, 8 mM ATP, 10 mM creatine phosphate) containing 1× protease and phosphatase inhibitor cocktail (Thermo Scientific, Waltham, MA, USA), and sonicated in chilled water using an ultrasound device (Bandelin, Berlin, Germany). Afterwards, the protein content of the cytosolic fraction was determined using the bicinchoninic acid assay (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s specifications.
Chymotrypsin-, trypsin-, and caspase-like proteasomal activities were measured using fluorogenic peptides, namely, Suc-LLVY-AMC (Sigma Aldrich, St. Louis, MO, USA), Bz-VGR-AMC (Enzo Life Sciences, Exeter, UK) and Z-LLG-AMC (Sigma Aldrich). The cytosolic proteins (20 µg) were incubated with reaction buffer (50 mM Tris, 0.5 mM EDTA) and 4 mM of each fluorogenic peptide at a time. Subsequently, the reaction was inhibited by adding 20 µM of proteasome inhibitor MG132 (Sigma Aldrich) which served as a negative control. The kinetic reaction was measured using a spectrophotometer (Tecan, Crailsheim, Germany) for 10 min at 380 nm excitatory and 440 nm emission wavelengths. For the calculation of the enzymatic activity, a calibration curve of free 7-amino-4-methylcoumarine was generated (Sigma Aldrich).

Pancreatic cancer cell lines were obtained from the American Type Culture Collection (ATCC) and cultured in the 5% CO₂ and 37 °C incubator in Roswell Park Memorial Institute medium supplemental with 10% FBS according to the ATCC recommendations. In some experiments, cells were treated 200 ng/ml of cytokines or chemokines and cultured for 48 h before harvesting. A431 or MCF7 cell lines with doxycycline-regulated expression of ZEB2 or ZEB1 were cultured in Dulbecco’s Modified Eagle’s medium supplemented with 10% foetal bovine serum (FBS) in the presence of absence of 1 μg/ml doxycycline. STAT3 inhibitor stattic (Sigma Aldrich, St Louis, MO, USA) was used at the concentration 5 μM. Proteasome inhibitor MG132 (Sigma Aldrich) was added at the increasing concentrations 16 h prior cell lysis. Cells were transfected by electroporation (a single pulse of 250V and 250 Fd by using the Gene Pulser Xcell electroporation system; BioRad Laboratories, Hercules, CA, USA) according to the manufacturer’s protocol.