Penicillin streptomycin

Caki-1, human renal cell carcinoma cell line, as well as HEK-293T, a human embryonic non-cancerous cell line, were generously donated by Dr. Dormond (Department of Visceral Surgery, CHUV, Lausanne, Switzerland). Caki-1 cells were cultured in DMEM medium and HEK-293T in RPMI medium supplemented with 10% fetal bovine serum (Biowest, Nuaillé, France, S1810-500) and 1% penicillin/streptomycin (Bioconcept, Basel, Switzerland, 4-01F00-H) in a humidified incubator with 5% CO₂ at 37 °C. ECRF24 cells [21 (link),65 (link)] were genuinely donated by Prof. Arjan W. Griffioen (Department of Medical Oncology, Vrije Universiteit Amsterdam, The Netherlands). ECRF24 cells were cultured in 0.2% gelatin-coated flasks in a 1:1 mixture of DMEM and RPMI medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. NHDFα cells were kept in a special fibroblast medium and supplements purchased from Vitaris (Baar, Switzerland, C-23120-PRO). Cells were tested frequently for mycoplasma contamination and were authenticated by Microsynth AG (Balgach, St. Gallen, Switzerland). Through STR systems from Promega (Zurich, Switzerland) and database comparison, the cell line identity was analyzed and confirmed.

The EBRTcH3 (EB3) parental mouse ES line and the clones overexpressing Sim2-Flag have been previously described in [24 (link), 25 (link)].
mES cells were grown on 0.1% gelatin (Sigma #G1890) coated dishes in DMEM high glucose medium (Life technologies #41965) supplemented with 15% Fetal Bovine Serum (FBS HyClone, Thermo Scientific #SH30070), 2mM L-glutamine (Life technologies #25030), 1mM Sodium pyruvate (Life technologies #11360), 100units/ml penicillin/streptomycin (Bioconcept #4-01F00-H), 0.1mM 2-mercaptoethanol (Life technologies #31350), 1000units/ml Leukemia Inhibitory Factor (LIF, Millipore #ESG1107) and 1μg/ml tetracycline (Sigma #T7660). Cells were incubated at 37°C in a 5% CO₂ atmosphere. Medium was changed every day and cells were passed every 1 or 2 days using 1X Trypsin-EDTA (Sigma #T4174).

Cryopreserved PBMC were stimulated with peptides (individual or peptide pools) at a final concentration of 1µg/mL per peptide in RPMI supplemented with 8% human serum (Biowest, Nuaillé, Maine-et-Loire, France), Penicillin/Streptomycin (BioConcept, Allschwil, Basel-Country, Switzerland) and beta-mercaptoethanol (Gibco). Recombinant human IL-2 (Proleukin, Clinigen, Yardley, Pennsylvania, United States) was added to the culture after 48 h at a final concentration of 100 U/mL for 12 days. At day 12 of the IVS, IFNγ Enzyme-Linked ImmunoSpot (ELISpot) assays were performed using pre-coated 96-well ELISpot plates (3420-2APT-10 Mabtech, Stockholm, Södermanland and Uppland, Sweden) [37 (link)]. Then, 100,000 cells were re-challenged (in duplicate) with peptides for 16–18 h, and then plates were washed according to manufacturer’s instructions and counted with the AID-Spot Robot ELISpot reader (AutoImmun Diagnostika GMBH, Straßberg, Baden-Württemberg, Germany). SEB (Staphylococcal Enterotoxin B, final concentration of 250ng/mL, Sigma-S4881) and anti-CD3 antibody (final concentration of 1 µg/mL, clone UCHT1, Mabtech) were used as positive control and unstimulated PBMCs as background control.

RPMI 1640 Glutamax Supplement (Thermo Fisher Scientific, Waltham, MA, United States), 10% Human Serum (BIOWEST, Riverside, MO, United States), 1mM Na pyruvate (Thermo Fisher Scientific, Waltham, MA, United States), 10mM/ml Hepes (Thermo Fisher Scientific, Waltham, MA, United States), 1X MEM Non-Essential Amino Acids (Thermo Fisher Scientific, Waltham, MA, United States), 0.1% β-mercaptoethanol and 1% penicillin-streptomycin (Bioconcept, Allschwil/BL, Switzerland).

The GT:ctgf, iS4, iS2 NIH-3T3 cell lines and HEK 293 T cells (ATCC) were cultured in DMEM (Thermofisher; 41966029), supplemented with 10% fetal bovine serum (Thermofisher, 10270106) and 1% penicillin/streptomycin (BioConcept, 4-01F00H), at 37 °C and 5% CO₂. Cells were grown in 100 mm cell culture plates up to a confluence of 70% and split 1/6 every 2–3 days.

All the in vitro studies were conducted in accordance with the local veterinary commission in Basel, Switzerland (No. 2925). Visceral adipose tissue was harvested from adult Sprague-Dawley rats and processed under sterile conditions as described earlier [21 (link)]. Briefly, the fat tissue was rinsed in 0.01 M phosphate-buffered solution (PBS), minced and resulting tissue was digested with 0.15% (w/v) Type I Collagenase (Gibco Life Technologies, Cat. No. 17100017) for 1 h at 37 °C and centrifuged for 5 min at 1500 rpm and 4 °C. The pellet was re-suspended in growth medium (GM) i.e., Dulbecco`s Modified Eagle`s Medium (DMEM, Gibco, Cat. No. 41965039) supplemented with 10% Foetal Bovine Serum (PAN-Biotech, EU-approved, Cat. No. P40-47500) and 1% Penicillin/Streptomycin (BioConcept, Cat. No. 4-01F00-H). Isolated ASC were seeded at a density of 3000 cells/cm² and expanded at 37 °C with 5% CO₂ in a humid atmosphere; GM was changed every 72 h. Cells were passaged using 0.25% Trypsin-EDTA (BioConcept, Cat. No. 5-51F00-H) at 90% confluence and resulting cells at passage 2 (P2) or 3 (P3) were used for the experiments.

HCC-derived cell lines (Hep3B (ATCC Number: HB-8064), Huh6 (JCRB Cell Bank Number: JCRB0401), SNU449 (ATCC Number: CRL-2234), SNU182 (ATCC Number: CRL-2235), SNU398 (ATCC Number: CRL-2233) and Huh7 (JCRB Cell Bank Number: JCRB0403)) and hepatoblastoma derived cell line HepG2 (ATCC Number: HB-8065) were maintained in a 5% CO2-humidified atmosphere at 37 °C and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin (Bio-Concept, Allschwil, Switzerland), and 1% minimal essential medium–nonessential amino acids (ThermoFisher Scientific, Basel, Switzerland). Cell lines were confirmed negative for mycoplasma infection using the polymerase chain reaction (PCR)-based Universal Mycoplasma Detection kit (American Type Culture Collection, Manassas, VA). For transient GPAM knockdown, log-phase HepG2 and Huh7 cells were seeded at approximately 60% confluence in 6-well plates and transfected with siRNA against human GPAM (Dharmacon, #L-009946-00-0005) or non-targeting control siRNA (Dharmacon, #D-001810-10-20) to a final concentration of 25 nM, according to the manufacturer’s protocol. Cells were harvested at 24, 48 and 72 h post transfection for protein isolation.