Epidermal growth factor (egf)

Mouse NSC line C17.2 [33 ] was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) containing 2 mM l-glutamine (Invitrogen), 10 % fetal bovine serum (FBS) (Sijiqing Biotech, China), 5 % horse serum (Gibco), 100 units/mL of penicillin, and 100 μg/mL of streptomycin. The cultures were maintained in a standard humidified incubator in 5 % CO₂ at 37 °C, with fresh medium replaced every 2 days, and split 1:4 when the cells reached 90 % confluence.
Primary neural stem cells were isolated from newborn SD rat cerebral cortex and cultured in uncoated 25-mL flasks in DMEM/F-12 medium (Invitrogen) containing N2 and B27 supplements (Invitrogen) plus basic fibroblast growth factor (Promega, 20 ng/mL) and epidermal growth factor (Promega, 20 ng/mL). After 5–7 days culture in vitro (DIV), the primary neurospheres were collected and dissociated with 0.05 % trypsin plus 200 mM EDTA for 10 min at 37 °C and mechanically triturated with fire-polished glass pipettes. The single cells were resuspended at a density of 50,000 cells per mL of serum-free medium and cultured for 3–5 days. The number and diameters of neurospheres were assessed as described [34 (link)].

The U251 cell line was obtained from ATCC (Manassas, VA, USA). GSC lines 387 and 3832 were generously provided by Dr Jeremy Rich, while all other GSC lines used were generated in-house from tissue samples obtained during surgical resection of patients diagnosed with GBM, using an IRB-approved protocol. Tumors were then subjected to enzymatic digest, mechanically dissociated and cultured as neurospheres as previously described by our group. Tumor lines were maintained as subcutaneous flank xenografts in athymic Nu/Nu mice and processed as stated above. All GSC lines were cultured in growth media made up of Neurobasal Media (Lifetech, Chicago, IL, USA) supplemented with N2 (Lifetech), GlutMAX (Lifetech), basic fibroblast growth factor and epidermal growth factor, both at 25 μg/ml (Promega Corp., Madison, WI, USA), unless stated otherwise. CBD was obtained from the National Institutes of Health through the National Institute of Drug Abuse. Ethanol served as the vehicle control in all culture studies. Tocopherol (TOC; Sigma-Aldrich, St. Louis, MO, USA), SAS (Sigma-Aldrich), Fer (Sigma-Aldrich), DFO (Abcam Ltd, Boston, MA, USA) and ERA (Selleck Chemicals, Houston, TX, USA) were all obtained commercially from indicated vendors. PE was generously donated by Dr. Brent Stockwell (Columbia University, New York, NY, USA).

Malignant gliomas are tumors characterized by high heterogeneity; therefore, our study used two intracranial glioma models, GL261-5 and GSC-005, to address the possibility of obtaining results due to the specific genetic makeup of a single model. The murine glioma cell line GL261-5 (a clone with slower in vivo growth kinetics compared with GL261, from the Tumor Bank Repository, National Cancer Institute, Frederick, MD, USA)²³ (link) was cultured in Dulbecco’s modified Eagle’s medium with nutrient mixture F12 (DMEM/F12) (Corning). Additionally, the murine GSC-005 glioma cells (kindly provided by I.M. Verma, The Salk Institute for Biological Studies, CA, USA)⁶¹ (link) were maintained in DMEM/F12 supplemented with N2 (1×, Invitrogen), fibroblast growth factor 2 (20 ng/mL, PeproTech), epidermal growth factor (20 ng/mL, Promega), and heparin (50 μg/mL, Sigma). Cell cultures were further supplemented with 10% fetal bovine serum (HyCline Laboratories), penicillin (100 μg/mL, Corning), and streptomycin (100 μg/mL, Corning) and were stored under the following conditions: 37°C and 5% CO₂.

Human microvascular endothelial cell line-1 (HMEC-1) was grown in MCDB131 medium (Gibco) supplemented with EGF (1 ng/mL; Promega) and hydrocortisone (1 ng/mL; Sigma). On the other hand, human umbilical vein endothelial cells (HUVECs) were grown on EBM-2 medium (Lonza), supplemented with SingleQuots (Lonza) endothelial growth medium-2 (EGM-2). All culture media were supplemented with 10% of FBS (Gibco), 2 mM of L-Glutamine and 100 units/mL of penicillin/estreptomicine (Gibco). Blood outgrowth endothelial cells (BOECs) from healthy and HHT donor primary cultures were grown in EGM-2 (Lonza), supplemented with 20% FBS, following the reported protocol.
43 (link)
44 (link)
Treatments in the absence or presence of FGF were performed in medium EGM with 2% FBS. BOECs from passages 4 and 5, three control groups, and three different HHT donors were used in the experiments.

Healthy and pSS glandular fragments belonging to each classification group were dissociated by enzymatic and mechanical means into a suspension of single cells and plated onto a culture flask. Cells were used to obtain the experimental material (RNA and proteins) used to carry out our evaluations. After dissociation pSS dispersal cells were resuspended in McCoy’s 5a modified medium supplemented with 10% heat-inactivated (56 °C for 30 min) FCS, 1% antibiotic solution, 2 mM L-glutamine, 50 ng mL⁻¹ epidermal growth factor (EGF, Promega, Madison, WI, USA), and 0.5 μg mL⁻¹ insulin (Novo, Bagsværd, Denmark) and incubated at 37 °C, 5% CO₂ in the air. The contaminating fibroblasts were selectively removed using 0.02% EDTA treatment. Immunocytochemical confirmation of the epithelial origin of cultured cells was routinely performed, as previously described [21 (link)]. Healthy human SGEC were grown in the same modified McCoy’s 5A medium (Invitrogen, Waltham, MA, USA) supplemented with 1% heat-inactivated FCS (to avoid excessive growth during treatment with TGF-β1). Healthy SGEC were stimulated with 10 ng/mL of TGF-β1 in the growth medium for 24–48 h and then harvested for FSTL1 analysis. To inhibit FSTL1, siRNA gene knockdown technique was used. All experiments were performed in triplicate and repeated three times.

The HT29 and HCT116 human colon adenocarcinoma cell lines were obtained from ATCC (American Type Culture Collection, Rockville, MD, USA). p53^+/+ and p53^−/− HCT116 cells were a kind gift of Bert Vogelstein and Kenneth W. Kinzler (Johns Hopkins University, Baltimore, USA). Cells were cultured at 37°C under a humidified atmosphere of 5 % CO2 in DMEM medium (GlutaMAX^TM, high glucose, Life Technologies, St Aubin, France) supplemented with 10 % heat inactivated-fetal calf serum (FCS) (PAN, Dominique Dutscher, Brumath, France). HIEC human healthy colon cells were a generous gift from Professor J.F. Beaulieu, University of Sherbrooke, Quebec, Canada. HIEC cells were cultured in OptiMEM-I (Life Technologies) supplemented with 5% FCS (Hyclone, Fisher Scientific, Illkirch, France), 0.01 M Hepes (Life Technologies) and 5 ng/ml EGF (Promega, Charbonnieres, France). Stable Bcl-2 transfected HT29 cells (HT29-Bcl2) and empty vector transfected HT29 cells (HT29-neo) were cultured as previously described [21 (link)].

Normal breast epithelial cell line MCF-10A, breast cancer cell lines MCF-7 and T47D were obtained from ATCC (Manassas, VA, USA). MCF-10A, MCF-7 and T47D cell lines were cultured in high-glucose (4.5 mg/ml) DMEM with 10% (v/v) FBS (HyClone). MCF-7 CSCs and T47D CSCs were induced and cultured in DMEM-F12 (Gibco, Thermo Fisher Scientific), containing 2% B27 (Gibco), b-FGF 10 μg/L (Promega), EGF 20 μg/L (Promega), as previously described [23 (link)]. All cells were maintained at 37 °C in a 5% CO₂ and 95% air incubator.