Leibovitz s l 15

HEY, A2780, CAOV3, ES-2 and SKOv3 human ovarian cancer cell lines were purchased from the American Type Culture Collection (Manassas, VA). EF021, EF027, OAW42, OC316 and IGROV1 were kindly provided by Dr. Gordon Mills’ laboratory [14 (link)–17 (link)] and all the cell lines were confirmed with STR DNA fingerprinting which was done by the MDACC Characterized Cell Line core (supported by NCI # CA016672). SKOv3 cells were culture with Macoy’s 5A; OC316, EFO27, EFO21, IGROV1, ES-2, A2780 and Hey cells were culture with RPMI1640; CAOV3 and OAW42 cells were cultured with DMEM. All media were obtained from the Media Preparation Core Facility at M. D. Anderson Cancer Center. SW626 cells were cultured with Leibovitz’s L-15 (Sigma-Aldrich, St. Louis, MO). All cell lines were tested for mycoplasma with a MycoSensor PCR Assay Kit from Stratagene (La Jolla, CA) and found to be free from contamination.

Zebrafish liver (ZFL) cell line was obtained from China Center for Type Culture Collection and maintained in medium containing 50% Leibovitz's L-15 (Sigma, # L1518), 35% Dulbecco's modified Eagle's medium (DMEM; GIBCO, # 12100046), and 15% Ham's F-12 (GIBCO, # 21700075) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO, # 10091148), 0.15 g/l sodium bicarbonate (GIBCO, # 25080094), 15 mM HEPES (GIBCO, # 15630080), 0.01 mg/ml insulin (GIBCO, # 41400045), 50 ng/ml epidermal growth factor (GIBCO, # PHG0311), and 2 mM GlutaMAX (GIBCO, # 35050061) at 28°C.
For proliferation detections, after 2 days of cells culture, cells were starved in serum-free medium for 12 h and changed to experimental medium (1% FBS, 0.15 g/l sodium bicarbonate, 15 mM HEPES, 0.01 mg/ml insulin, 50 ng/ml epidermal growth factor, and 2 mM GlutaMAX) with 1, 2, 5, 8, 10 μg/ml PNA for 48 h before lysis. Cells without lectin treatment were served as the control. All experiments were repeated at least three times.

The zebrafish liver cell line (CVCL_3276) (Ghosh et al. 1994 (link)) was purchased from ATCC (Mannassas, USA). A nutrition medium consisting of 50% (v/v) Leibovitz’s L-15 (Sigma-Aldrich, Steinheim, Germany), 35% (v/v) Dulbecco’s modified Eagle’s medium (Gibco, Paisley, UK), 15% (v/v) Ham’s Nutrient Mixture F-12 (Gibco, Paisley, UK), and phenol red, supplemented by 150 mg/L sodium bicarbonate (Gibco, Paisley, UK), 15 mM HEPES (Gibco, Paisley, UK), 10 μg/mL bovine insulin (Sigma-Aldrich, Steinheim, Germany), 50 ng/mL mouse EGF (Sigma-Aldrich, Steinheim, Germany), and 5% (v/v) fetal bovine serum (Gibco, Paisley, UK), was used for cultivation. The cells were cultured in a humidified environment at 28 °C and atmospheric CO₂. Further, cells were passaged weekly in a 1:20 subcultivation ratio. Phosphate buffered saline (PBS, pH 7.4; Medicago, Uppsala, Sweden) was used for washing and 0.25% (w/v) trypsin–EDTA (Sigma-Aldrich, Steinheim, Germany) for cell detachment. All experiments were conducted within passage numbers 5 to 32.

Cancer cell lines were purchased from ATCC except for Huh7 (JCRB), PC-9 (Riken BRC), BEL7404 (SUZHOU BEILE BIOTECH), SK-OV-3, A2780, RPMI-8226 (ECACC), HCC78 (DSMZ) and KM-12 (HUATUO). Cell culture medium included RPMI-1640 (#22400-089), McCoy’s 5a (#16600082), DMEM (#11995-065), F-12K (#21127-022) and IMDM (#12440053) was purchased from Gibco. EMEM (#30-2003) and Hybri-Care (#46-X) medium was purchased from ATCC. Leibovitz’s L-15 (#L1518) was purchased from SIGMA. All the medium was contained 10% or 20% FBS (#FND500, ExCell Bio). The 2D and 3D models used the same culture medium. Horse serum (#041241A) was bought from BI. Trypsin (#25200072) and antibiotic-antimycotic (#15240-062) was purchased from Gibco. Dulbecco’s PBS (#21-031-CVC) and matrigel (#354234) were purchased from Corning. DMSO (#D2650) was bought from Sigma. CellTiter-Glo Cell Viability Assay kit (#G7573) and CellTiter-Glo 3D Cell Viability Assay kit (#G9683) were purchased from Promega.

Aag2 and S2 cells were a kind gift from Raul Andino (University of California, San Francisco, USA) and were maintained at 28°C in a humidified atmosphere without CO₂ as previously described [27 (link)]. Culture medium was Leibovitz’s L-15 (Sigma-Aldrich, St. Louis, MO USA) supplemented with 10% (v/v) tryptose phosphate broth (Sigma-Aldrich), 10% (v/v) USA-origin foetal bovine serum (FBS), 2 mM L-glutamine, 0.1 mM non-essential amino acids, and 100 U/ml penicillin/100 μg/ml streptomycin (all ThermoFisher Scientific, Waltham, MA USA).

Zebrafish liver cell line ZFL^{26 (link),27 (link)} (CVCL_3276) was cultured in a medium consisting of 50% Leibovitz’s L-15 (Sigma-Aldrich, Steinheim, Germany), 35% Dulbecco’s modified Eagle’s medium (Gibco, Paisley, UK), 15% Ham’s Nutrient Mixture F-12 (Gibco, Paisley, UK), and phenol red. Additionally, 150 mg/L sodium bicarbonate (Gibco, Paisley, UK), 15 mM HEPES (Gibco, Paisley, UK), 10 µg/mL bovine insulin (Sigma-Aldrich, Steinheim, Germany), 50 ng/mL mouse EGF (Sigma-Aldrich, Steinheim, Germany), and 5% fetal bovine serum (Gibco, Paisley, UK) were supplemented. The cells were cultured in a humidified environment at 28 °C and atmospheric CO₂. The cells were passaged every 3 to 4 days in a 1 to 4 ratio, using PBS (Medicago, Uppsala, Sweden) for washing and 0.25% Trypsin-EDTA (Sigma-Aldrich, Steinheim, Germany) for detachment.

Cell culture was performed as described previously^{37 (link)}. A2058 cells were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). SKMEL28 cells were obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Japan. A2058 and SKMEL28 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, UT, USA). WM3211 cells were obtained from the Rockland Immunochemicals Inc (Limerick, PA, USA) and maintained in a culture medium consisting of 80% MCDB153 (Sigma) with 20% Leibovitz’s L-15 (Sigma), 2% FBS (HyClone), and 1.68-mM CaCl₂ (Sigma). All cells were cultured at 37 °C in a humidified incubator under 5% CO₂. FLAG-PITX1 stable expression A2058 clones were maintained in DMEM supplemented with 10% FBS and G418 (300 μg/ml; Calbiochem, La Jolla, CA, USA). All cell lines were confirmed to be mycoplasma-free using a MycoAlert mycoplasma detection kit (Lonza, Walkersville, MD, USA) and were not passaged more than 20 times from the validated stocks. Fluorescent images were obtained using a KEYENCE BZ-X710 microscope (Keyence, Osaka, Japan) using an objective with a 40 × magnification.