Nms 873

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).

P22077 (Merck-Millipore), NMS873 (Tocris), cycloheximide (Sigma, Merck), ML792 (Synthetized in the CNIO) and MLN7243 (Chemietek) were dissolved in DMSO; cells were incubated for the indicated time in the presence of the inhibitor or an equivalent amount of DMSO. Whole cell extracts were prepared by lysing cells in 50 mM Tris, pH 7.5, 8 M Urea, and 1% Chaps. Cytosolic and nuclear extracts were prepared following the protocol described before (Lecona et al., 2008 (link)) and the chromatin fraction was then extracted in 50 mM Tris, pH 7.5, 8 M Urea, and 1% Chaps. Transfection of RPE cells with specific siRNA was carried out using Lipofectamine RNAimax (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s instructions and using pools of 4 specific siRNA directed against the indicated proteins (Dharmacon, Horizon Discovery). Transfection of HCT116 cells with the pCL-His-hUbi plasmid (Young et al., 2011 (link)) was carried out using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific) following the manufacturer’s instructions.