A172, LN18, U87MG, and U373 GBM cell lines and human embryonic kidney 293 T cells were obtained from American Tissue Type Culture Collection (USA) and cultured in DMEM medium (Gibco, USA) with %10 fetal bovine serum (Gibco, USA) and %1 penicillin-streptomycin (Gibco, USA). MGG152, GBM8-TR, and GBM8-TS cells were kindly gifted by Dr. Hiroaki Wakimoto (Massachusetts General Hospital, Boston, MA)19 (link). GBM8-TR cells were cultured in DMEM (Gibco) supplemented with 10% FBS and 1% penicillin/streptomycin. GBM8-TS cells were cultured in neurobasal medium (Gibco) supplemented with 3 mmol/L of L-Glutamine (Mediatech), B27 (Invitrogen/GIBCO), 2 μg/mL of heparin (StemCell Technologies), 20 ng/mL of human EGF (R&D Systems), and 20 ng/mL of human FGF-2 (fibroblast growth factor; PeproTech). All cells were grown in 37 °C, 5% CO2 in a humidified incubator. TRAIL was commercially supplied (Enzo Life Sciences, US) or produced from 293T cells as described29 (link). Stock of MS-275 (Cayman Chemicals, USA) was prepared in DMSO. ZVAD-FmK and ZVA-FMK were prepared in DMSO (Promega, USA).
Human egf
Manufactured by R&D Systems
Sourced in United States
Human EGF is a recombinant human epidermal growth factor. It is a potent mitogen that stimulates the growth of a variety of cell types. Human EGF promotes cell proliferation, differentiation, and survival.
Automatically generated - may contain errors
Lab products found in correlation
Neurobasal medium, by Thermo Fisher Scientific (5 mentions)
Heparin, by Merck Group (4 mentions)
Human fgf2, by Thermo Fisher Scientific (4 mentions)
N2 supplement, by Thermo Fisher Scientific (4 mentions)
B27 supplement, by Thermo Fisher Scientific (4 mentions)
Human bmp4, by R&D Systems (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (3 mentions)
Human lif, by Merck Group (3 mentions)
Dmem medium, by Thermo Fisher Scientific (2 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (2 mentions)
Human plasma fibronectin, by Thermo Fisher Scientific (2 mentions)
Activin a, by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (2 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
96 well u bottom plate, by Corning (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
24 protocols using human egf
1
Culturing Glioblastoma and Kidney Cell Lines
2
Cell Lines Characterization for Research
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MDA-MB231 cells were kindly provided by Dr. Chiara Riganti (Department of Oncology, University of Turin, Torino, Italy). MDA-MB231, U138 MG, and T47-D cells were purchased from ATCC and were cultured in DMEM/F12 medium (Gibco BRL, Karlsruhe, Germany). GBM20 cells were a kind gift from Prof. R. Glass (Munich, Germany) and were kept in DMEM/F12 medium supplemented with 1× B 27, 1% v/v penicillin-streptomycin, 10 ng/mL human EGF (R&D Systems, Minneapolis, MN, USA) and 10 ng/mL human FGF-basic (PeproTech, Hamburg, Germany). All other cell lines were grown in RPMI 1640 medium supplemented with 8 % v/v fetal bovine serum, 1% v/v L-Glutamine, and 1 % v/v penicillin-streptomycin). All cells were incubated in a humidified CO2 incubator at 37 °C, 18.3% O2 and 5% CO2. Cell line identification was performed by STR-PCR (Eurofins, Ebersberg, Germany).
3
Induction of PGCLC from Dissociated Cells
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For PGCLC induction, cells were dissociated by incubation with Accutase and then transferred into a low-cell-binding U-bottom 96-well plate (Greiner, #650970) in GK15 medium (GMEM (Thermo, #11017035) containing 15% KSR, 0.1 mM NEAA, 1 mM sodium pyruvate (Thermo, #11360070), 2 mM GlutaMax, 0.1 mM 2-mercaptoethanol, and 1 × penicillin/streptomycin) supplemented with 200 ng/ml human BMP4 (R&D Systems, #314-BP-01 M), 100 ng/ml mouse SCF (R&D Systems, #455-MC-500), 1000 U/ml human LIF (Wako, #125-06661), 50 ng/ml human EGF (R&D Systems, #236-EG) and 10 μM ROCK inhibitor with or without 2.5 μM IWR1 (TOCRIS, #3532) and 1.0 µM BMS493 (R&D Systems, #3509/10). In each well, 5 × 103 cells were used. In the experiments requiring enforced expression of the transgenes, 100 μM dexamethasone, 0.5 μg/ml doxycycline, and 0.5 μM Shield-1, which induce SOX17, BLIMP1, and TFAP2C expression, respectively, were added to the medium11 (link). To enforce the expression of SOX17 alone, only 100 μM dexamethasone was added.
4
Glioblastoma Cell Culture Protocol
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A172 and U373, LN18, LN229, T98G and U87MG GBM cell lines and human embryonic kidney 293T cells were obtained from American Tissue Type Culture Collection (Manassas, VA, USA) and cultured in DMEM medium (Gibco, Gaithersburg, MD, USA) with 10% fetal bovine serum and 1% Penicillin-Streptomycin (Gibco, Gaithersburg, MD, USA). GBM8, GBM4 and MGG119 cells [19 (link),34 (link)] were cultured in neurobasal medium (Gibco, Gaithersburg, MD, USA) supplemented with 3 mM L-Glutamine (Mediatech/Sigma-Aldrich, Woburn, MA, USA), B27 (Invitrogen/Gibco, Norcross, GA, USA), 2 μg/mL heparin (StemCell Technologies/Fisher Scientific, Kent, WA, USA), 20 ng/mL human EGF (R&D Systems, Minneapolis, MN, USA), and 20 ng/mL human FGF-2 (PeproTech Hamburg, Germany) (EF media). All cells were grown in 37 °C, 5% CO2 in a humidified incubator. Vitronectin (Gibco, Gaithersburg, MD, USA), Collagen (Gibco, Gaithersburg, MD, USA), recombinant human TGFβ1 (Peprotech 100-21, Hamburg, Germany), Tiplaxtinin (Selleckchem PAI-039, Houston, TX, USA), Repsox (Tocris, Ellisville, MO, USA), and SB431542 (Stemcell Technologies, Kent, WA, USA) were used for dispersal experiments. D-luciferin was used for in vivo imaging (Biotium, Fremont, CA, USA).
5
hESCs to human PGCLCs Differentiation
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hESCs were dissociated into single cells with 0.05% Trypsin-EDTA (GIBCO, 25300-054) and plated onto Human Plasma Fibronectin (Invitrogen, 33016-015)-coated 12-well-plates at the density of 200,000 cells/well in 2mL/well of iMeLC media, which is composed of 15% KSR (GIBCO, 10828-028), 1x NEAA (GIBCO, 11140-050), 0.1mM 2-Mercaptoethanol (GIBCO, 21985-023), 1x Penicillin-Strep-tomycin-Glutamine (GIBCO, 10378-016), 1mM sodium pyruvate (GIBCO, 11360-070), 50ng/mL Activin A (Peprotech, AF-120-14E), 3mM CHIR99021 (Stemgent,04-0004), 10mM of ROCKi (Y27632, Stemgent, 04-0012-10), and 50ng/mL primocin in Glasgow’s MEM (GMEM) (GIBCO, 11710-035). After 24 hr, iMeLCs were dissociated into single cells with 0.05% Trypsin-EDTA and plated into ultralow cell attachment U-bottom 96-well plates (Corning, 7007) at the density of 3,000 cells/well in 200ml/well of hPGCLC media, which is composed of 15% KSR (GIBCO, 10828-028), 1x NEAA (GIBCO, 11140-050), 0.1mM 2-Mercaptoethanol (GIBCO, 21985-023), 1x Penicillin-Streptomycin-Glutamine (GIBCO, 10378-016), 1mM sodium pyruvate (GIBCO, 11360-070), 10ng/mL human LIF (Millipore, LIF1005), 200ng/mL human BMP4 (R&D systems, 314-BP), 50ng/mL human EGF (R&D systems, 236-EG), 10mM of ROCKi (Y27632, Stemgent, 04-0012-10), and 50ng/mL primocin in Glasgow’s MEM (GMEM) (GIBCO, 11710-035).
6
Osteosarcoma Cell Line Culturing and Stimulation
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Osteosarcoma cell line 143B, MG63, U2OS were purchased from American Type Culture Collection (Manassas, VA, USA). Well5 was established by our group [47 (link)]. All cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen), supplemented with 10% fetal bovine serum (FBS, Invitrogen), penicillin (100 U/ml) and streptomycin (100 mg/ml; Invitrogen), at 37°C in a 5% CO2 incubator.
For continuous exposure to human TSP1 (R&D Systems) or human FGF-2 (R&D Systems) or human VEGF (Peprotech) or human EGF (R&D Systems) or human TGF-β1 (Peprotech), cells were seeded on culture dishes that had been coated with TSP1 (2 ug/ml) or FGF-2(10 ng/ml) or EGF (20 ng/ml) or VEGF (5 ng/ml) or TGF-β1(5 ng/ml) for 12h.
For continuous exposure to human TSP1 (R&D Systems) or human FGF-2 (R&D Systems) or human VEGF (Peprotech) or human EGF (R&D Systems) or human TGF-β1 (Peprotech), cells were seeded on culture dishes that had been coated with TSP1 (2 ug/ml) or FGF-2(10 ng/ml) or EGF (20 ng/ml) or VEGF (5 ng/ml) or TGF-β1(5 ng/ml) for 12h.
7
EGFR ECD and EGF Interaction with NE
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human EGFR ECD (500 ng; R&D Systems) and human EGF (500 ng; R&D Systems) were treated with multiple doses of NE (100–300 mU/ml) in the presence or the absence of NE inhibitor, sivelestat sodium hydrate (100 μg/ml; ONO Pharmaceutical Co) at 37 °C for 3 h. Samples were separated by Tris–glycine–SDS-PAGE using 12% polyacrylamide gels (Bio-Rad) or tricine–SDS-PAGE with a 16% Peptide-PAGE mini (TEFCO). The gels were stained with Coomassie Brilliant Blue (Apro Science).
8
Investigating Tumor Cell Growth Inhibition
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The effect of various inhibitors on tumor cell growth and signaling was assessed as described previously.13 (link) Cell growth between treatment groups was compared by one-way analysis of variance with Tukey's multiple comparison test (groups were confirmed to have similar variances). Cells were treated with the following reagents (doses and treatment times are indicated in the Figure Legends): REGN1400 (ERBB3-blocking antibody), REGN955 (EGFR-blocking antibody), MEK inhibitor GSK1120212 (Selleckchem, Houston, TX, USA), FGFR TKI AZD4547 (Selleckchem), MET TKI PHA665752 (Sigma, St Louis, MO, USA), EGFR TKI AZD9291 (Selleckchem), PI3K inhibitor BYL719 (Selleckchem), human NRG1 (R&D Systems, Minneapolis, MN, USA) and human EGF (R&D Systems). The following antibodies were used for western blot analyses: ERBB3 (CST, Danvers, MA, USA, cat. #4754), phospho-ERBB3 (CST, cat. #4561), EGFR (CST, cat. #2646), phospho-EGFR (CST, cat. #2234), Akt (CST, cat. #9272), phospho-Akt (CST, cat. #4060), ERK (CST, cat. #4695), phospho-ERK (CST, cat. #4370), MET (CST, cat. #8198), phospho-MET (CST, cat. #3077) and phospho-FGFR (CST, cat. #3476).
The tyrosine phosphorylation status of 49 human RTKs was assessed using the Human Phospho-RTK Array Kit (R&D Systems) as described previously.
The tyrosine phosphorylation status of 49 human RTKs was assessed using the Human Phospho-RTK Array Kit (R&D Systems) as described previously.
9
Glioma Cell Culture Conditions
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CT2A and GL261 were cultured in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO) supplemented with 10% fetal calf serum (Valley Biomedical Inc.) and 1% penicillin/streptomycin (Invitrogen). 005-GFP, Mut3 and Mut4 cells were cultured in 1:1 neurobasal medium (GIBCO) supplemented with 1% penicillin/streptomycin (Invitrogen), 1% N2 supplement, 2 µg/ml heparin (sigma), B27(Invitrogen/ GIBCO), 20 ng/mL of human EGF (R&D Systems), and 20 ng/mL of human FGF-2 (fibroblast growth factor; PeproTech).
10
Isolation and Culture of Neural Stem Cells
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Rats were sacrificed by CO2 asphyxiation and neural stem cells were isolated from 13.5 days embryos of Wistar rats and cultured in growth medium with the 1% N2 (Gibco), 10 ng/ml bFGF (R&D), and 20 ng/ml human EGF (R&D) supplement. Primary neurospheres were digested using 0.25% trypsin to derive clone neurospheres. miR–381 mimic and scramble were purchased from Ambion. The transfection of miR–381 mimics and scramble (20ng/ml), Hes–1 vector and related controls was performed using Lipofectamine 2000 (Invitrogen) following to the manufacturer’s instruction.
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