Hcc1937

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).

MDA-MB-231, MCF-7 and HCC1937 cells were received from the Korea Cell Line Bank (Seoul, Republic of Korea). 4T1 cell were obtained from American Type Culture Collection (ATCC). MDA-MB-231 and HCC1937 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Thermo Fisher Scientific Inc., Waltham, MA, USA). 4T1 and MC38 cells were purchased from Kerafast Inc. (Boston, MA, USA). 4T1 cells were cultured in Dulbecco’s modified eagle’s medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Thermo Fisher Scientific Inc., USA). MC38 cells were cultured in Dulbecco’s modified eagle’s medium supplemented with 10% FBS, 2 mM glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 10 mM Hepes, 25 µg/mL gentamycin sulfate, and 1% penicillin/streptomycin (Thermo Fisher Scientific Inc.). Cells were incubated at 37 °C in an atmosphere of 5% CO₂. Cell culture ware, including cell culture dishes and plates, was purchased from SPL Life Sciences Co. (Pocheon-si, Gyeonggi-do, South Korea).

SK-BR-3, T47D, MDA-MB-468, and MDA-MB-157 cell lines were purchased and authenticated from the American Type Culture Collection (ATCC^®, Manassas, VA, USA). MCF7, ZR-75-1, BT-20, HCC-1937, and MDA-MB-231 were purchased and authenticated from the Korean Cell Line Bank (KCLB^®, Seoul, Republic of Korea). Human breast cancer cells were grown in DMEM (SK-BR-3, MDA-MB-157, MDA-MB-468, and Hs578T) or RPMI (MCF7, T47D, ZR-75-1, BT-20, HCC-1937, and MDA-MB-231) with 10% fetal bovine serum (FBS) (Gibco, Waltham, MA, USA) and 0.2% MycoZap™ Plus-CL (Lonza, Portsmouth, NH, USA) in a 37 °C humidified incubator with a 5% CO₂ atmosphere. Cells were seeded on 10 cm dishes for RNAi transfection. Two ATP1A1 siRNAs and scrambled siRNA were purchased from Ambion and transfected into seeded cells using Lipofectamine RNAiMAX (Invitrogen, Waltham, MA, USA). For plasmid transfection, the ATP1A1 CDS was cloned into the pCMV-Tag2B vector and transfected into the cells using FuGene (Promega, Madison, WI, USA). Cells were harvested for further analysis and other experiments.