Ham s f 12 medium

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.

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)].

HuCCT-1, a human CCA cell line, was obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank, Osaka, Japan. RMCCA-1, a human CCA cell line, was developed from Thai patients with CCA^{70 (link)}. Both HuCCT-1 and RMCCA-1 were cultured in HAM’s F-12 medium (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). Jurkat, a human T cell line, was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Jurkat was cultured in RPMI (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). All cell lines were cultured in a humidified incubator at 37 °C with 5% CO₂. All cell lines were tested for mycoplasma contamination and were mycoplasma free.