Ac anw amc

MDA-MB-231, HCC1143, and HCC1937 breast cancer cells were purchased from the Korean Cell Line Bank (Seoul, Korea). Hep3B, Huh7, LCSC, HepG2, and PLC/PRF/5 hepatic cancer cells were a kind gift of Dr. Roberto Gedaly (College of Medicine, University of Kentucky). All other established cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD). All cells were cultured according to the manufacturer’s protocol in 5% CO₂ in medium. Cultured cell lines were tested for Mycoplasma contamination routinely every 6 months. Specifically, H23 and H727 cells were tested twice in the course of performing the experiments described within this publication (Supplementary Fig. S4). Inhibitors of UPS pathways used in this study were purchased from commercial vendors: carfilzomib (LC Laboratories, Woburn, MA), bortezomib (ChemieTek, Indianapolis, IN), MG-132 (EMD Millipore, San Diego, CA), PYR-41 (ApexBio, Houston, TX), and P5091 (ApexBio, Houston, TX). The following proteasome fluorogenic substrates were used: Suc-LLVY-AMC (Bachem, Torrance, CA; I-1395), Ac-WLA-AMC (Boston Biochem, Cambridge, MA; S-330), Ac-nLPnLD-AMC (Bachem; I-1850), Ac-RLR-AMC (Boston Biochem; S-290), Ac-ANW-AMC (Boston Biochem; S-320), and Ac-PAL-AMC (Boston Biochem; S-310). Human recombinant Interferon-γ was purchased from eBioscience (San Diego, CA).

The chymotrypsin-like and beta5i-specific proteasome activities were determined in cellular lysates similarly as described in [23 (link)]. In brief, two fluorogenic substrates Suc-LLVY-AMC (Sigma, St. Louis, MO, USA) and Ac-ANW-AMC (Boston Biochem, Cambridge, MA, USA) were used to estimate the chymotrypsin-like and β5i-specific proteasome activities, correspondingly. Aliquots (~6 µL) of lysates were incubated in 100 μL of the reaction buffer (RB), containing 40 mM Tris-HCl (pH 7.5), 1 mM DTT, 5 mM MgCl₂, 1 mM ATP, and 30 μM of substrate for 20 min at 37 °C. Control reactions with 100 nM of the proteasome inhibitor Bortezomib were performed to test nonspecific degradation of substrates. Reactions were stopped with 2% SDS solution (in ddH2O). Fluorescence at the excitation wavelength 380 nm and emission wavelength 440 nm was measured using VersaFluor Fluorometer (Bio-Rad, Hercules, CA, USA). To calculate the relative activity levels, the activity levels in samples with Bortezomib were subtracted from the values detected in lysates and the obtained values were normalized to one µg of total protein. Proteasome activity in living cells was determined using cell-permeable proteasome activity probe Me4BodipyFL-Ahx3Leu3VS (UbiQbio, Amsterdam, The Netherlands) according to the protocol described in [24 (link)].