M199 medium

Promastigote form L. mexicana (WHO strain MNYC/BZ/62/M379), L. major Friedlin and their genetically modified derivatives were grown at 28°C in M199 medium (Life Technologies) supplemented with 2.2 g l⁻¹ NaHCO₃, 0.005% haemin, 40 mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid pH 7.4 and 10% FCS. Relevant selection drugs (Melford Laboratories Ltd.) were added to the medium at the following concentrations: 32 µg ml⁻¹ Hygromycin B, 20 µg ml⁻¹ Puromycin Dihydrochloride, 5 µg ml⁻¹ Blasticidin S Hydrochloride, 40 µg ml⁻¹ G-418 Disulfate, 50 µg ml⁻¹ Nourseothricin Sulfate and 25 µg ml⁻¹ Phleomycin. Trypanosoma brucei SmOx B4 [49 (link)] was grown in HMI-9 [54 (link)] at 37°C in 5% CO₂, T. brucei SmOx P9 in SDM-79 [55 (link)] at 28°C. Relevant selection drugs (Melford Laboratories Ltd.) were added to the medium at the following concentrations: 0.2 µg ml⁻¹ Puromycin Dihydrochloride, 5 µg ml⁻¹ Blasticidin S Hydrochloride, 5 µg ml⁻¹ G-418 Disulfate, 5 µg/ml Hygromycin B. Cell culture densities were measured using a CASY model TT cell counter (Roche Diagnostics) with a 60 µm capillary and exclusion of particles with a pseudo diameter below 2.0 µm.

Porcine ovaries from non-cycling gilts were collected at a commercial slaughterhouse (Jatky Český Brod a.s., Český Brod, CR) and transported in physiological saline at 37 °C to the laboratory. Cumulus-oocyte complexes were aspirated from follicles and matured in M199 medium (Life technologies, Carlsbad, CA, U.S.A.) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St-Louis, MO, U.S.A.) and 0.8 IU/mL P.G. 600 (Intervet). IVM was performed at 38.5 °C in a humidified atmosphere of 5% CO₂ for 12 to 44 h. Oocytes were denuded, washed and stored at −80 °C until use. For evaluation of maturation, denuded oocytes were fixed in ethanol:acetic acid solution (3:1 v/v) for 48 h. Staining was performed with 1% orcein in 50% aqueous acetic acid and 1% sodium citrate followed by washing with 40% acetic acid. Oocytes were observed and photographed under a phase-contrast microscope (Carl Zeiss, Jena, Germany).

Clone 9 rat hepatocytes, N1-S1 rat HCC cells, and HUVECs were purchased from the Food Industry Research and Development Institute. HepG2, Hep3B, PLC, Huh7, Mahlavu, and SK-Hep-1 Human HCC cells were purchased from the American Type Culture Collection with validated STR-PCR profile. Clone 9 cells cultured in F12-K medium (Gibco) containing 10% fetal calf serum (HyClone). N1S1 cells were maintained in RPMI-1640 medium (Gibco) containing 10% calf serum (HyClone). HepG2, Hep3B, PLC, Huh7, Mahlavu, and SK-Hep-1 human hepatoma cells were cultured in Dulbecco's modified Eagle's medium (Gibco) containing 10% calf serum (HyClone). HUVECs (passage: 3–6) were cultured in M199 medium (Life Technologies) containing 15% fetal calf serum, 20 U/ml porcine heparin (Sigma), and 100 g/ml endothelial cell growth supplement (Calbiochem). All the media for cell culture were supplemented with 2 mM L-glutamine (HyClone), 100 mg/ml streptomycin (HyClone), and 100 U/ml penicillin (HyClone). All cells were maintained under humidified conditions in 95% air and 5% CO₂ at 37 °C. Recombinant HGF (H5791), sodium acetate, lithium acetate (a GSK3β inhibitor) (46 (link)) and quercetin (β-catenin inhibitor) (47 (link)) were purchased from Sigma-Aldrich. XL184 (c-MET inhibitor) (48 (link)) was purchased from TargetMol.

M199 medium, Rowell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Life Technologies, Inc. (Grand Island, NY, USA). The RNA extraction kit was purchased from Intron Biotechnology (Seoul, Republic of Korea). FMN1, ITGA2, COL13A1, VEGFC, NRG1, and β-actin oligonucleotide primers and ITGA2 and VEGFC siRNA were purchased from Bioneer Technology (Seoul, Republic of Korea). 2-mercaptoethanol and phorbol myristate acetate (PMA) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). CCR2 antagonist RS102895 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). CCR5 antagonist fuscin (3,4,7,9-Tetrahydroxy-6-methyl-1H-phenalen-1-one) was purchased from Adipogen Life Sciences (San Diego, CA, USA). JNK inhibitor SP600125, PI3K/Akt inhibitor LY294002, and JAK2/STAT3 inhibitor AG490 were obtained from Calbiochem (San Diego, CA, USA). Dimethyl sulfoxide (DMSO) and CellTracker^TM were obtained from Invitrogen (Grand Island, NY, USA). Recombinant human CC chemokine ligand 2 (CCL2) and CCL5 were purchased from Thermo Fisher Scientific (Waltham, MA, USA).