Geneticin g418

B16F1 melanoma cells were transfected with the plasmids containing human SNAIL. The pcDNA 3.1-human SNAIL construct was obtained from Prof. Muh-Hwa Yang (Taipei Veterans General Hospital, Taiwan). B16F1 melanoma cells were grown up to 85% confluence in 1 g/L glucose DMEM Biosera LTD (Courtaboeuf CEDEX, France) in presence of 0.1% penicillin/streptomycin and 10% FCS at 37 °C with 5% CO₂. The Amaxa Nucleofector X Unit (Lonza, Basel, Switzerland) was used for the transfection of cells with 5 μg DNA/10⁶ cells, according to the manufacturer’s instructions. Cells were cultured in medium with additional 200 μg/mL of Geneticin/G418 (Gibco/LifeTechnologies, Waltham, MA, USA). After two weeks of cell culture and refreshing selection media every two days, the colonies, which were well-separated, were selected. The culture of clones was scaled-up and SNAIL expression was verified by RT-PCR and Western blot analysis. Mock-B16F1 cells, transfected with an empty plasmid, cultured with media supplemented with G418 (200 μg/mL), were used as controls.

Normal thyroid samples were obtained from patients undergoing surgery at IRCCS Istituto Nazionale dei Tumori (Milan, Italy). All patients gave their written informed consent, and the study was approved by the Independent Ethical Committee of Fondazione IRCCS Istituto Nazionale dei Tumori. Primary thyrocyte cultures were established and maintained in nutrient mixture Ham's F12 medium (custom made by Invitrogen, Carlsbad, CA, USA) containing 5% calf serum and bovine hypothalamus and pituitary extracts, as previously described [40 (link)].
4-hydroxytamoxifen (4OHT) (Sigma Aldrich, St Louis, Mo, USA) was used at 200 nM to activate RAS oncogene. Geneticin (G418, GIBCO, Carlsbad, CA, USA) and puromycin (Invitrogen, Carlsbad, CA, USA) were used for cell selection at 400 μg/ml and 2 μg/ml, respectively.

Human epithelial cell HEK-293T (ATCC^® CRL-3216™), simian epithelial cell Vero E6 (ATCC^® CRL-1586™), human astrocytoma cell U-87MG (ATCC^® HTB-14™), and human neuroblastoma cell SH-SY5Y (ATCC^® CRL-2266™) were provided by CelluloNet (SFR-Biosciences, Lyon, France). HEK-293T, Vero E6, and U-87MG cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; GlutaMAX, high glucose, Gibco™, Thermo Fisher Scientific, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco™). SH-SY5Y cells were grown in DMEM/nutrient mixture F-12 (DMEM/F-12, Ham 1:1, GlutaMAX, Gibco™) supplemented with 2% heat-inactivated FBS. Cell cultures were maintained at 37 °C and 5% CO₂. For the production of recombinant sNS1 protein, geneticin™-G418 (500 μg/mL, Gibco™) was added to the culture medium of HEK-293T cells 3 days before seeding them for transfection.
The IS-98-ST1 strain of WNV (provided by Dr. S. Lecollinet; ANSES-ENVA, Maisons-Alfort, France) is a highly virulent strain isolated from a stork with severe neurological symptoms during the 1998 epidemic in Israel [62 (link)]. Working stocks of WNV IS-98-ST1 were generated by a single round of amplification in Vero E6 cells. Titers of virus stocks were determined by a standard serial dilution plaque assay in Vero E6 cells, and results expressed as PFU/mL [1 (link),63 (link)].

Lentiviral construct coding for LMNA R321X mCherry-tagged protein was produced by Stratagene's Quik Change II XL site-directed mutagenesis kit (Agilent Technologies), using the the lentiviral plasmid encoding for mCherry-tagged Lamin WT mCherry-tagged (pLV[Exp]-Neo-CMV > mCherry(ns):hLMNA[NM_170707.4], Vector Builder). Mutagenic primers were designed using Quik Change Primer Design Program as previously shown [17 (link)]. The mutation was verified by sequencing.
Viral particles were produced in HEK-293T cells by simultaneously co-transfection (Invitrogen™ Lipofectamine™ 2000 Transfection Reagent) of the plasmid carrying the gene for protein of interest (mCherry-tagged-Lamin WT or mCherry-tagged-LMNA R321X), the envelope plasmid encoding VSV-G and the packaging plasmid encoding Gag/Pol and Rev, as previously reported [13 (link)].
For lentiviral transduction, HL-1 cells were plated in a 6-well plate at 30–50% of confluency, and after adhesion, they were incubated with 500 µL of virus-containing medium at 37 °C in a humidified 5% CO₂ incubator for 18 h. After viral particles removal, cells were then incubated with 400 μg/mL Geneticin (G418, Gibco, Life Technologies) for 1 week to select stable clones. Pure clones were selected by fluorescence microscopy.