William s e medium

The GBC lines used in this study were as follows: GBC-SD, SGC-996 were purchased from Shanghai Institute for Biological Science, Chinese Academy of Science (Shanghai, China). NOZ, OCUG-1, EHGB-1, EHGB-2 were purchased from the Health Science Research Resources Bank (Osaka, Japan). GBC-SD, EHGB-1, EHGB-2, OCUG-1 cells were cultured separately in DMEM (Gibco) with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin–streptomycin (Gibco). NOZ cells were cultured in William’s medium E (Lonza) with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin–streptomycin (Gibco). Contrastingly, SGC-996 cells were cultured in RPMI 1640 medium (Hyclone) with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin–streptomycin (Gibco). All of above cells were cultured in their respective media in a humidified incubator at 37 °C with 5% CO₂.

This study was reviewed and approved by the ethics committee of Xinhua Hospital, School of Medicine, Shanghai Jiaotong University. Written informed consent was obtained from all of the patients enrolled in this study. GBC tissue specimens were obtained from 74 patients who underwent radical cholecystectomy (without prior radiotherapy or chemotherapy) from 2004 to 2012 in the Department of General Surgery, Xinhua Hospital. Additionally, 60 patients with chronic cholecystitis who underwent simple cholecystectomy were included as controls. The tumor stage for the GBC participants was defined according to the 7th AJCC-TNM classification system. The present GBC study population included 21 males and 53 females with a mean age of 66 years (range 39–86 years). All patients were periodically followed up for survival data until July 2014. The human GBC cell lines NOZ (K-ras mutant) and SGC-996 (K-ras wild-type) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) [5 (link)]. The NOZ cells were cultured in William’s medium E (Lonza, Belgium, WI), and the SGC-996 cells were maintained in RPMI 1640 medium (Gibco, Gaithersburg, MD) at 37°C in a humidified 5 % CO₂ incubator. Both media were supplemented with 10 % fetal bovine serum (FBS). PD0325901 was obtained from Selleckchem (Houston, TX, USA).

The chicken hepatocellular carcinoma cell line LMH was cultured on gelatin-coated flasks in William’s Medium E (Lonza) supplemented with 2 mM glutamine and 10% fetal bovine serum (FBS) at 37 °C in a 5% CO2 atmosphere. Chicken embryonic skin cells (CESCs) were prepared from 12-old day specific pathogen-free LD1 embryo and cultivated as previously described [33 (link)].
The pEGFP-UL49 MDV plasmid encoding MDV VP22 tagged with EGFP at its N-terminal extremity was previously described [11 (link)]. The genes encoding the PRV UL49, ILTV UL49 and VZV UL49/ORF9 were amplified from the PTD12 [9 (link)], pcDNA-ILTV49 [39 (link)] and pEGFP-9p plasmids, respectively (kindly provided by Pr L. Enquist, Dr W. Fuchs, Pr C. Sadzot). It is worth noting, for the sake of clarity in this manuscript, the terms VZV UL49/ORF9 and VZV VP22/ORF9p will be used instead of ORF9 and ORF9p alone.

Mouse hepatocytes were prepared and used in experiments have been described previously [25 (link)]. Briefly, the hepatocytes were isolated by two-step collagenase perfusion from 6 different strains of mouse; NMRI, SV129, FVB, DBA, BALB/C and C57BL/6. The mouse strains differed with respect to age (2–6 months) and gender (female or male) [25 (link)]. Cells were plated on collagen-coated six-well plates. Cell viability was determined by Trypan Blue exclusion, and was at least 75%. Hepatocytes obtained for 6 different mouse strains were cultured independently in William’s E medium (Lonza Bioscience, Verviers, Belgium) supplemented with 10% (v/v) fetal calf serum, 20 m-units/mL insulin, 10 nM dexamethasone, 100 U/mL penicillin, 100 mg/mL of streptomycin, 0.25 mg/mL fungizone and 50 mg/mL gentamycin. After four hours the medium was discarded and replaced with fresh medium. The next day, cells were incubated in fresh medium in the presence or absence of Wy14643 (10 μM) dissolved in dimethyl sulfoxide (DMSO) for 6 and 24 h, followed by RNA isolation. Isolation of mouse primary hepatocytes was approved by the Ethical Committee for Animal Experiments of Wageningen University.

Placentas were obtained from women who delivered at term (38 ± 2 gestational weeks). All participants provided written informed consent prior to the collection of placentas. The collection and use of placentas were approved by the Institutional Review Board of the Gangnam CHA General Hospital, Seoul, Korea (07–18). CP-MSCs were harvested as previously described [27 (link)]. Briefly, CP-MSCs were harvested from the inner side of the chorion-amniotic plate of the placenta by treatment with 0.5% collagenase IV (Sigma-Aldrich) for 30 min at 37 °C. The cells harvested from the placenta were cultured in Ham’s F-12/Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) with 1% P/S, (Invitrogen), 10% fetal bovine serum (FBS, Invitrogen), 1 µg/mL heparin (Sigma-Aldrich), and 25 ng/mL FGF-4 (Peprotech, Inc., Rocky Hill, NJ, USA). The WI-38 cells (ATCC, Manassas, VA, USA) were cultured with alpha-MEM (Invitrogen) supplemented with 1% P/S and 10% FBS. Isolated primary rat hepatocytes were cultured with William’s E medium (Lonza, Basel, Switzerland) supplemented with 10% FBS and 2% P/S at 37 °C.