Penicillin streptomycin solution

Human hepatocellular carcinoma HuH-7 cells (a kind gift from Dr Colin Adrain, Instituto Gulbenkian de Ciência, Portugal), Human Embryonic Kidney 293T (provided by Prof Paul Digard, Roslin Institute, UK) and 293ET cells (from Dr Colin Adrain) were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, 21969035) supplemented with 10% fetal bovine serum (FBS, Gibco, 10500064), 1% penicillin/streptomycin solution (Biowest, L0022) and 2mM L-glutamine (ThermoFisher, 25030024), at 37°C and 5% CO₂ atmosphere. Plasmids used in this study were obtained as follows: pLEX.MSC was purchased from Thermo Scientific; psPAX2 and pVSV.G were a kind gift from Dr Luís Moita (Instituto Gulbenkian de Ciência, Portugal); pEGFP-N1 was provided by Dr Colin Crump (University of Cambridge, UK); and pCAGGS containing the SARS-Related Coronavirus 2, Wuhan-Hu-1 Spike Glycoprotein Gene (pCAGGS-SARS-CoV-2-S, NR-5231) was obtained through BEI Resources.

Human colon cancer cells Caco-2 obtained from ATCC (Rockville, MD, USA) were propagated in Eagle’s Minimum Essential Medium (EMEM) obtained from Biowest (Nuaille, FR) supplemented with 10% fetal bovine serum, 1% sodium bicarbonate, 1% sodium pyruvate, 5 mM glutamine, 1% Minimum Essential Medium nonessential amino acids, and 1% penicillin-streptomycin solution (10,000 units/mL of penicillin and 10 mg/mL of streptomycin). The components for cells’ cultivation were purchased from Sigma-Aldrich. Cultures were incubated at 37 °C in a humidified atmosphere of 5% CO₂ in the air.

Isolation and cultivation of the rabbit BMSCs were accomplished by institute of Hansen’s disease, Catholic university. Animal experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) in College of Medicine, the Catholic University of Korea (CUMC-2010-0034-01). Briefly, bone marrow cells were aspirated from the femur and tibia of 4 weeks old New Zealand White rabbits (Orient Bio Inc., Seongnam, Korea) and re-suspended in phosphate-buffered saline (PBS; Biowest, Nuaillé, France). The Collected cells were slowly layered above FiColl-Paque PLUS (GE Healthcare, Amersham Biosciences, UK), for gradient centrifugation at 400×g for 30 min. After centrifugation, bone marrow cells were separated over the gradient interface. These cells are aspirated and rinsed with PBS. Subsequently, these bone marrow cells were cultured with low glucose Dulbecco's Modified Eagles Medium (DMEM; Biowest, Nuaillé, France) containing 10% fetal bovine serum (FBS; Biowest, Nuaillé, France) and 1% penicillin streptomycin solution (Biowest, Nuaillé, France) at 37 °C in humidified atmospheres with 5% CO₂. After overnight incubation, the non-adherent cells were removed by replacing the medium. Thereafter, the culture medium was replaced every 3 days. At 80-90% confluence, cells were sub-cultured to passage 7 by using culture medium.

Chitosan (the low molecular weight, deacetylation degree of 79%), phosphate-buffered saline (PBS; KH₂PO₄, K₂HPO₄) (pH 7.4), human serum albumin (HSA), 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), reagents for Winkler method, glycerin and diiodomethane (pure for analysis), methanol gradient grade were purchased from Sigma-Aldrich (Munich, Germany). Water was purified with a Milli-Q Water Purification System (Millipore Corp., Bedford, MA). 20% Cannabis oil (CBD Pure) was purchased from a local pharmacy.
All reagents for the fibroblast cell culture: Eagle's Minimum Essential Medium (EMEM), fetal bovine serum (FBS), Non-Essential Amino Acids (NEAA), L-glutamine, trypsin–EDTA, penicillin/streptomycin solution, Dulbecco’s phosphate buffer saline (DPBS) were purchased from Biowest (Nuaillé, France). 2-Propanol and hydrochloric acid (pure for analysis) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; suitable for cell culture), were purchased from Sigma Aldrich.

Primary antibodies: anti-phospho-FGFR (Tyr653/Tyr654) (p-FGFR) (#06-1433) were from Merck (Darmstadt, Germany); anti-phospho-mTOR (Ser2448) (p-mTOR) (#2971), anti-FGFR1 (FGFR1) (#9740), anti-phospho-EGFR (Y1173) (p-EGFR) (#4407), anti-phospho-AKT (Ser473) (p-AKT p-S473) (#9271), anti-phospho-AKT (Thr308) (p-AKT p-T308) (#9275), anti-AKT1/2/3 (AKT) (#9272), anti-phospho-p44/42 (Thr202/Tyr204) MAP kinase (p-ERK1/2) (#9101), anti-p44/42 MAP kinase (ERK1/2) (#9102), anti-MDR1/ABCB1 (MDR1) (#12683), and anti-poly-[ADP-ribose] polymerase (PARP) (#9542) were from Cell Signaling Technology (Danvers, MA, USA); anti-mTOR (mTOR) (#T2949), anti-γ-tubulin (tubulin) (#T6557) and anti-acetylated-α-tubulin (ac-tubulin) (#T7451) were from Sigma Aldrich (St Louis, MO, USA). Horseradish peroxidase-conjugated secondary antibodies were from Jackson Immuno-Research Laboratories (Cambridge, UK) and a chemiluminescent substrate was used to visualize them in the ChemiDoc station (BioRad, Hercules, CA, USA). AlexaFluor^®594-conjugated secondary antibodies were from Abcam (Cambridge, UK). NucBlue Live ReadyProbes Reagent was from Thermo Fisher (Waltham, MA, USA). Geneticin (G-418) was from BioShop (Puck, Poland). Penicillin-Streptomycin Solution was from Biowest (Nuaille, France). Heparin came from Sigma-Aldrich.

RPMI 1640 medium and DMEM (Dulbecco’s Modified Eagle Medium) were acquired from Capricorn Scientific GmbH (Ebsdorfergrund, Germany). L-glutamine, trypsin, and penicillin–streptomycin solution were from Biowest (Nuaillé, France). Thiazolyl blue tetrazolium bromide (MTT), fetal bovine serum (FBS), DMSO, Hoechst 33342, and carboxyfluorescein succinimidyl ester (CFSE) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). An Apoptosis Detection Kit based on Annexin-V-FITC (AV) and propidium iodide (PI) staining was acquired from Abcam (Cambridge, UK). Bovine serum albumin (BSA) was obtained from Serva (Heidelberg, Germany). Rhodamine 123 (Rho123) and tetramethylrhodamine ethyl ester (TMRE) were purchased from Sigma (St Louis, MO, USA). Carbonyl cyanide m-chlorophenyl hydrazine (CCCP, C2759) was purchased from Sigma-Aldrich (Darmstadt, Germany). FITC-conjugated anti-P-gp antibody was obtained from BD Biosciences (Winnersh, Berkshire, UK). Anti-P-gp mouse monoclonal antibody (Abcam, Cambridge, UK; ab10333) and secondary antibodies Alexa Fluor 555 goat anti-mouse secondary antibody (#A-21422, Thermo Fisher Scientific, Waltham, MA, USA) were obtained.