Kku 214

Human CCA cell lines (KKU213, KKU100, KKU214, KKU-M055, HuCCT-1) and a nontumor human cholangiocyte cell line (MMNK1) were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank, Osaka, Japan. RMCCA-1 cells were developed from Thai patients with CCA [56 (link)]. All human CCA cell lines and MMNK1 were cultured in HAM’s F-12 medium (HyClone Laboratories, Logan, Utah, USA). All culture media were supplemented with 10% fetal bovine serum (Sigma, St Louis, Missouri, USA) and 1% Penicillin-Streptomycin (HyClone Laboratories, Logan, Utah, USA). All cells were cultured in a humidified incubator at 37 °C with 5% CO₂. All cell lines were tested for mycoplasma contamination and were mycoplasma free. For drug treatment, cells were pretreated with Smac mimetic, SM-164 (5 nM for KKU213 or 50 nM for KKU100, HuCCT-1 and MMNK1) or Smac mimetic and zVAD-fmk (20 μM) for 2 h, after that cells were transfected with 2.5 μg/ml Poly(I:C) by TurboFect transfection reagent (Thermo fisher scientific, Waltham, Massachusetts, USA). Combination index (CI) for Poly(I:C) and Smac mimetic combination treatment was calculated based on Chou-Talalay method using CompuSyn version 1.0 software [57 (link)].

Human CCA cell lines; namely KKU-213 and KKU-214 were established from CCA patients and obtained from Japanese Collection of Research Bioresources (JCRB) Cell Bank, Osaka, Japan. All cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco/Invitrogen, Calsbald, CA) with normal (N; 5.56 mM) or high (H; 25 mM) concentrations of glucose supplemented with 10% fetal bovine serum (Gibco/Invitrogen) and a 1% antibiotic-antimycotic (Gibco/Invitrogen). Cells were incubated in a 37 °C, 5% CO₂, humidified incubator. All cell lines were cultured in N or H media for at least 5 passages allowing for their adaptation prior to use48 (link). Cells harvested from the culture conditions for normal glucose media were designated as NG cells and those cultured in high glucose media were designated as HG cells.
Paraffin-embedded histologically proven CCA tissues (n = 20) were obtained from the specimen bank of the Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Thailand. Written informed consent was obtained from each subject and the protocol has been reviewed and approved by The Khon Kaen University Ethics Committee for Human Research (HE571464) based on the Declaration of Helsinki and ICH-Good Clinical Practice Guidelines. The experiments were conducted in accordance with the approved protocols.

CCA cell lines (KKU213, KKU100, KKU214, KKU-M055, HuCCT-1) and MMNK1 were provided from the Japanese Collection of Research Bioresources (JCRB) Cell Bank, Osaka, Japan. HuCCA-1 [39 (link)] and RMCCA-1 [40 (link)] were developed from Thai patients with CCA. HT-29 and HEK293T were obtained from American Type Culture Collection (ATCC). All CCA cell lines and MMNK1 were grown in HAM's F-12 medium (HyClone Laboratories, Logan, Utah, USA), while HT-29 and HEK293T were cultured in Dulbecco's modification of Eagle's medium (DMEM; HyClone Laboratories, Logan, Utah, USA) supplemented with 10% fetal bovine serum (Sigma, St Louis, Missouri, USA) and 1% penicillin streptomycin (HyClone Laboratories, Logan, Utah, USA) under the standard protocol at 37˚C in 5% CO₂ humidified atmosphere. All cultures were tested for mycoplasma contamination and were mycoplasma-free.

CCA cell lines (KKU‐100, KKU‐213, and KKU‐214) were obtained from the Japanese Collection of Research Bioresources (JCBR) Cell Bank (Osaka, Japan). MMNK1, an immortal cholangiocyte cell line, was a gift from Kobayashi N. (Maruyama et al., 2004). Cells were cultured in DMEM—Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% FBS and 1% antibiotic–antimycotic under standard protocol. Transient enhancement of O‐GlcNAcylation was performed by culturing cells in the presence of 20 μm PUGNAc for 24 h prior to further experiments.
The immunohistochemistry (IHC) experiments were performed using formalin‐fixed paraffin‐embed liver tissues from histologically proven CCA patients. Each subject gave informed consent, and the study protocol was certified by the Ethics Committee for Human Research at Khon Kaen University (HE581369).

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).