F 12 nutrient mixture ham s f 12

HeLa (human cervical cancer-derived) cells, Chinese hamster ovary (CHO)-K1 cells, and CHO-A745 cells (lacking xylosyltransferase), were purchased from the Riken BRC Cell Bank (Ibaraki, Japan) (HeLa cells) and American Type Culture Collection (Manassas, VA, USA) (CHO-K1 and CHO-A745 cells), respectively. Each cell was cultured in α-MEM (Gibco, Life Technologies Corporation, Grand Island, NY, USA) (HeLa cells) and F-12 nutrient mixture (Ham’s F-12) (CHO-K1 and CHO-A745 cells) (Gibco, Life Technologies Corporation) containing 10% heat-inactivated FBS (Gibco, Life Technologies Corporation). Each cell was grown on 100-mm dishes and incubated at 37 °C under 5% CO₂.

CCA cell lines (KKU-213 and KKU-214), were obtained from the Japanese Collection of Research Bioresources (JCBR) Cell Bank, Osaka, Japan. The cell lines were cultured in F-12 Nutrient Mixture (Ham’s F-12) (Gibco, NY) containing 10% FBS and 1% antibiotic-antimycotic. Transient enhancement of O-GlcNAcylation was performed using OGA inhibitor [O-(2-Acetamido-2-deoxy-D-glucopyranosylidenamino)-N-phenylcarbamate; PUGNAc]. Cells were incubated with 20 μM PUGNAc (Sigma Aldrich, St. Louis, MO.) for 1 h before subjecting them to further experiments. Inhibition of nuclear translocation of NF-κB was employed by incubating cells with 2.5 μg/ml dehydroxymethylepoxyquinomicin (DHMEQ)21 (link).

Fmoc‐amino acids and coupling reagents were purchased from either Anaspec (Fremont, CA) or Novabiochem (Germany). PEG500 (NH₂‐(PEG)‐COOH, 0.5 kDa), PEG2000 (2 kDa), and PEG5000 (5 kDa) were purchased from Nektar Therapeutics (Huntsville, AL). G7 PAMAM dendrimers were purchased from Dendritech Inc. (Midland, MI). CellTracker Green and PBS solution were obtained from Thermo Fisher Scientific (Waltham, MA). Fetal bovine serum (FBS) and antibiotic mixture (penicillin/streptomycin) were acquired from Invitrogen. Dulbecco's modification of Eagle's medium (DMEM), DMEM/Ham's F‐12 50/50 mix (DMEM/F‐12), and Roswell Park Memorial Institute (RPMI) medium were purchased from Corning (Manassas, VA). F‐12 nutrient mixture (Ham's F12) and trypsin‐EDTA were obtained from Gibco (Grand Island, NY). Recombinant human EpCAM was purchased from R&D Systems. All other chemicals were obtained from Sigma‐Aldrich (St. Louis, MO), unless otherwise stated.