Eeyarestatin 1

HEK293, HeLa, PC3, SW480, SW620, HCT116, HCC1419, HT29, MDAMB231, A549, MG63, MCF7, and BT474 (ATCC, VA, USA) cells were cultured in Dulbecco's modified Eagle medium (DMEM; Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin and streptomycin (Gibco) at 37 °C in a humidified atmosphere with 5% CO₂. Cycloheximide, MG132, and Chloroquine were purchased from Sigma-Aldrich (MO, USA). YM155 was purchased from Selleckchem (TX, USA) and Eeyarestatin I was purchased from Tocris. Flag-SLC35F2 was received from Dr. Giulio Superti-Furga. Flag-SLC35F2 was then subcloned into a pcDNA 3.1 6XMyc-vector. HA-USP32 WT and HA-USP32 CA were received from Dr. Jacques Neefjes. HA-Ubiquitin (Addgene #18712) was procured from Addgene (MA, USA). Cas9-2A-mRFP-2A-PAC was purchased from Toolgen (Seoul, South Korea).

Human EC cell lines (KYSE70, poorly differentiated invasive esophageal squamous cell carcinoma; KYSE140, moderately differentiated invasive esophageal squamous cell carcinoma) were purchased from the Type Culture Collection of the Chinese Academy of Sciences. SHEE cell line (an immortalized epithelium of the fetal esophageal epithelium) was obtained from Prof. Liu (China–US Hormel Cancer Institute). All the cells were maintained at 37°C with a humidified atmosphere of 5% CO₂ and cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin–streptomycin liquid. ERAD inhibitors including Eeyarestatin I (known as EerI, #3929, US) and NMS-873 (#6180, US) were purchased from Tocris Bioscience (UK).