Human recombinant epidermal growth factor egf

HK2 (CRL-2190™, ATCC, Rockville, MD, USA), HeLa, and HEK293  cell lines were grown in 25 cm² flasks in a 1 : 1 mixture of Dulbecco's modified Eagle medium (DMEM GIBCO-BRL® Life Tech, Thermo Fisher, MA, USA), Ham's F₁₂ nutrient mixture for HK2 cells plus 10% fetal bovine serum (FBS, GIBCO-BRL®), and DMEM medium for HEK293 and HeLa cells plus 5% FBS. The medium was supplemented with 2 mM L-glutamine (Sigma-Aldrich, MO, USA), 15 mM HEPES (Sigma-Aldrich), and 5 ng/ml recombinant human epidermal growth factor (EGF, Life Technologies) only for HK2 cells (pH 7.4), and gentamicin was used for HEK293. The cultures were incubated at 37°C in a 5% CO₂ atmosphere and 95% of relative humidity until cell monolayers were formed. The medium was replaced every 72 h, and when monolayers reached at least 80% of confluence, they were washed with a free Ca²⁺ and Mg²⁺ saline phosphate buffer (PBS: 138 mM NaCl, 3 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.4) and incubated with 0.125% trypsin and 0.5 mM EDTA solution for 15 min, and in some HEK293 cultures, Hanks solution was used for detaching cells. Detached cells were harvested and suspended in fresh medium at a concentration of 10⁶ cells/ml to subculture in 24-well microplates for experiments.

Gold (III) chloride trihydrate (HAuCl₄·3H₂O), hyaluronic acid (HA) sodium salt from Streptococcus equi (MW 7000–250,000 g/mol), oleic acid (OA) (MW 282.46 g/mol), trypsin, fetal bovine serum (FBS), and penicillin/streptomycin were all supplied from Sigma-Aldrich (Steinheim, Germany). Dulbecco’s modified Eagle medium (DMEM) was supplied by Biowest (Nuaillé, France). The human keratinocyte cell line, HaCat, the human melanoma cell line, A375, and the murine melanoma cell line, B16F10, were provided by Cell Line Service GmbH (Eppelheim, Germany). Recombinant human epidermal growth factor (EGF) was purchased from Life Technologies (Waltham, MA, USA). The water used in all the experiments was purified through a Millipore system (Millipore, Burlington, MA, USA). All the remaining chemicals and substrates used were of analytical grade.
Intramuscular administration of a mixture of ketamine (Ketamidor^®, Richter Pharma, Wels, Austria) and chlorpromazine (Largactil^®, Laboratórios Vitória, S.A., Amadora, Portugal) (10:2 v/v) at a dose of 2 mL/kg was used as anesthetic protocol for the in vivo experiments.

For the propagation of rNSC, the culture medium (complete StemPro NSC SFM) was similar to the one used in our earlier publication [46 (link)]. It is composed of recombinant human Fibroblast Growth Factor basic (FGFb, 20 ng/mL, Gibco, Life Technologies Corporation, Carlsbad, CA, USA ), recombinant human Epidermal Growth Factor (EGF, 20 ng/mL, Gibco, Life Technologies Corporation, Carlsbad, CA, USA), Glutamax (2 mM 100×), and StemPro Neural Supplement (2% in 1× KnockOut D-MEM/F-12 medium, Gibco, Life Technologies Corporation, Carlsbad, CA, USA). T75 culture flask was coated with 15 mL of Poly-L-ornithine (20 μg/mL, Sigma, Burlington, MA, USA) and incubated overnight at room temperature (RT), rinsed twice with D-PBS (in the absence of Ca²⁺ and Mg²⁺ and used for the rNSC propagations. Multipotent fetal rNSC (Gibco) were subjected to 3-types of the directed-differentiation process to obtained astrocytes, neurons, and oligodendrocytes. The rNSCs were cultured (2 × 10⁶ cells) in the stem cell growth medium in the incubator at 37 °C, 5% CO₂, and 90% humidity. The following day, the medium was replaced with a pre-warmed stem cell growth medium. The growth medium was changed with a fresh growth medium every 2–3 days, and the cells were passaged when confluency reached 75–90%. Passage number 3 rNSCs (P3) were used for the differentiation experiments [46 (link)].

The primary cultured GSCs SU4 and SU5 were derived from fresh surgical specimen of human GBM tissues using a method described previously with informed consent [37 (link)]. The study was approved by the Ethics Committee of the Second Affiliated Hospital of Soochow University. Cell lines were maintained in Dulbecco’s modified Eagle’s medium/F12 (Gibco, Grand Island, NY, USA) supplemented with B-27 (Gibco, Grand Island, NY, USA), 20 ng/mL recombinant human epidermal growth factor (EGF; Gibco, Grand Island, NY, USA), 20 ng/mL basic fibroblast growth factor (bFGF; Gibco, Grand Island, NY, USA) and antibiotics (100 U/mL penicillin and 100 mg/L streptomycin (HyClone, South Logan, UT, USA)).
TMZ, Nicardipine, chloroquine (CQ), rapamycin, laminine and poly-L-ornithine solutions were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Dimethyl sulfoxide was the product of MP Biomedicals, LLC (Santa Ana, CA, USA). Antibodies against β-actin, Bcl-2, Bax, p62, LC3B, AKT, p-AKT, mTOR and p-mTOR, were purchased from Cell Signaling Technology (Danvers, MA, USA). The secondary biotinylated antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).

Ethylene glycol, ferric chloride, and sodium acetate were obtained from Aladdin (Shanghai, China). Sodium hydroxide and tetraethoxysilane (TEOS) were obtained from Macklin (Shanghai, China). Laminin and acrylamide were obtained from Sigma-Aldrich (MO, United States). Phosphate buffered saline (PBS), accutase, Hanks’ balanced salt solution (HBSS), B-27, and recombinant human epidermal growth factor (EGF) were obtained from Gibco (NY, United States). Recombinant murine FGF-basic (FGF) was obtained from PropTech (NJ, United States). The NeuroCult™ differentiation kit was obtained from STEMCELL Technologies (CA, United States). The nestin marker (Nestin antibody, AN205), TuJ-1 marker (neuronal class III β-tubulin, AT809), and DAPI (C1002) were obtained from Beyotime (Jiangsu, China). Donkey anti-mouse secondary antibody (A21202) was obtained from ThermoFisher (MA, United States).

CSC cultures using SKOV3, SKOV3-RFP, and SKOV3-ARID3B cells, were generated by treating cells with 20μM of cisplatin (Sigma, St. Louis, MO) for 72h. Surviving cells were trypsinized and cultured in serum free DMEM/F12 media (Life Technologies, Grand Island, NY) supplemented 5μg/ml insulin (Invitrogen, Carlsbad, USA), 10 ng/ml human recombinant epidermal growth factor (EGF; Invitrogen, Carlsbad, USA), 10 ng/ml basic fibroblast growth factor (bFGF, Invitrogen, Carlsbad, USA), 12 ng/ml leukemia inhibitory factor (LIF; Santa Cruz Biotechnologies, Santa Cruz, CA) and 0.4% bovine serum albumin (BSA; Sigma, St. Louis, MO). The selected cells formed non-adherent spheres when grown in this condition. The media was changed every 2 days. SKOV3 CSCs were collected for RNA analysis and flow cytometry.

Trichomonas vaginalis isolate T016 isolate was kindly provided by Prof. John F. Alderete (School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, WA, USA) [29 (link)] and grown in Diamond’s trypticase-yeast extract-maltose (TYM) medium supplemented with 10% heat-inactivated horse serum (Invitrogen, Carlsbad, CA, USA) at 37°C [30 ]. The human normal prostatic epithelial cell line (RWPE-1) was obtained from the American Type Culture Collection (ATCC; CRL-11609) and cultured in keratinocyte serum-free medium (K-SFM) supplemented with 25 μg/ml bovine pituitary extract (BPE), 5 ng/ml human recombinant epidermal growth factor (EGF), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA, USA). The human monocytic leukemia cell line (THP-1) and human prostate cancer cell lines (PC3, DU145, and LNCaP) were purchased from the ATCC (Manassas, VA, USA). Cell lines were maintained in RPMI1640 (Hyclone, Logan, UT, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) and 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in 5% CO₂.