G418 disulfate salt solution

Human pharyngeal SCC cell line FaDu (ATCC, HTB-43) was grown in Advanced Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Thermo Fisher, MA, USA) supplemented with 5% fetal bovine serum (FBS, Gibco, Thermo Fisher), 10 mM L-glutamine (GlutaMAX, Gibco), penicillin (100 U/mL) (Grünenthal, Germany) and gentamicin (50 mg/mL) (Krka, Slovenia). Cells were routinely subcultured twice a week and incubated in a humidified atmosphere at 37 °C and 5% CO₂.
The HPV-positive 2A3 cell line, a kind gift from Prof. Dadachova was established by transfection of FaDu cells with HPV16 oncogenes E6 and E7 [12 (link)]. 2A3 cells were grown in Advanced DMEM supplemented with 5% fetal bovine serum, 10 mM L-glutamine, penicillin (100 U/mL), gentamicin (50 mg/mL) and 1 mg/mL G418 disulfate salt solution (Sigma-Aldrich, MO, USA).
Authentication of FaDu, FaDu-RR, and 2A3 cells by short tandem repeats profiling was performed using CellCheck 16 – human (IDEXX BioAnalytics, Germany) for authentication of FaDu, and FaDu-RR cells (Additional file 1: Final report of laboratory examination) and CellCheck 9 – human (IDEXX BioAnalytics) for 2A3 cells (Additional file 2: Final report of laboratory examination). The genetic profile of the cell lines used for the study was identical to the publically available genetic profile of these cell lines.

The promoter-trap CHO-K1 monitoring cell line generated in a previous study contained a promoter-less EGFP expression cassette [23] (link). A total of 1.0 × 10⁶ cells of monitoring cell lines were transfected with 5 μg of sgRNA1 sgRNA-Cas9-mCherry and 5 μg of each promoter HDR KI donor plasmid using a NEPA21 electroporator for site-specific targeted integration. After 3 d, transfected cells were seeded at 0.3 × 10⁶ cells/mL in suspension 6-well cell culture plate with 3 mL PowerCHO-2CD medium supplemented with 8 mM L-glutamine and 500 μg/mL G418 disulfate salt solution (G8168, Sigma Aldrich). EGFP expression levels were determined using flow cytometry 6 d after transfection.

Hexarelin, Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (DME/F-12), G418 disulfate salt solution, hydrogen peroxide (H₂O₂), 3 (4,5 dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT), poly-D-lysine hydrobromide, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), fluoromount aqueous mounting medium, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Penicillin, streptomycin, L-glutamine, trypsin-EDTA, phosphate-buffer saline (PBS) and fetal bovine serum (FBS) were obtained from Euroclone (Pero, Milan, Italy). Alexa Fluor 488 Phalloidin was purchased from ThermoFisher Scientific (Waltham, MA, USA).
The JMV2894 was synthetized by conventional solid phase from the laboratory of Professor Jean-Alain Fehrentz, Institut des Biolécules Max Mousseron, University of Montpellier (France). Compounds were purified by high performance liquid chromatography (HPLC) (purity ≥ 98%).
Prior to assay, the GHSs were freshly dissolved in ultrapure water. Both GHS and H₂O₂ were diluted in culture media to final working concentrations for the experiments.

HepG2 cells (ATCC‐HB‐8065 from ATCC, Manassas, VA, USA) were stably transfected with a human oncostatin M cDNA in order to overexpress OSM (H/OSM) or with the empty vector (pCMV6) alone (H/V6) and selected with G418 disulfate salt solution (Merck‐Sigma Aldrich, St Louis, MO, USA) as described previously [26 (link)]. HepG2 naïve cells and HepG2 transgenic cells maintained in Dulbecco's modified Eagle's medium low glucose (DMEM, 1000 mg/l, from Merck‐Sigma Aldrich) were seeded in normoxic conditions to obtain the desired sub‐confluence level (65–70%) and for performing the subsequent experimental analyses.
Human umbilical vein endothelial cells (HUVECs) were isolated, maintained, and used as pools of five different donors to minimize cell variability as previously described [27 (link)]. Collection of umbilical cords for isolation of HUVECs was under an agreement between the University of Torino and the Azienda Ospedaliera Ordine Mauriziano di Torino Hospital, protocol number 1431 02/09/2014. Informed consent was obtained from all subjects involved.

All cell lines were cultured at 37 °C in a 5% CO₂ humidified atmosphere. The pharyngeal SCC cell line FaDu (ATCC®, Gaithersburg, MD) was cultured in RPMI 1460 medium (Thermo Fisher Scientific Inc., Rockford, IL). The HPV E6 and E7 viral protein-expressing cell line 2A3 (derived from the FaDu cell line, a gift from Prof. Dadachova^{18 (link)}) was cultured in Dulbecco’s Minimum Essential Medium (Thermo Fisher Scientific) supplemented with 1 mg mL⁻¹ G418 disulfate salt solution (Sigma-Aldrich LLC, St. Louis, MO). Both media were supplemented with 5% fetal bovine serum (Thermo Fisher Scientific), 10 mM L-glutamine (Thermo Fisher Scientific), 100 U/mL penicillin (Grünenthal, Aachen, Germany) and 50 mg/mL gentamicin (KRKA, Novo Mesto, Slovenia). The methods used to determine the presence and expression of the HPV16 E7 gene in the 2A3 cell line and the corresponding results are described in Prevc et al. 2018, Supplementary Materials^{6 (link)}.