The human prostate epithelial cell line RWPE-1 (ATCC) was cultured in keratinocyte serum free medium (Invitrogen) supplemented with 50 μg/ml bovine pituitary extract and 5 ng/ml recombinant human epidermal growth factor (rhEGF). The human mammary epithelial cell line184 A1N4 (a gift from M. R. Stampfer, UC Berkeley) [23 (link)] was cultured in modified Improved Minimum Essential Medium (IMEM) supplemented with 0.5% FBS, 5 μg/ml recombinant human insulin (rh-insulin) (Invitrogen), 5 μg/ml hydrocortisone (Sigma), and 10 ng/ml rhEGF (Promega). The milk-derived human mammary epithelial cell lines MTSV-1.1 B and MTSV-1.7 (a gift from Dr. J. Taylor-Papadimitriou, Imperial Cancer Research Fund, London) [24 (link)] were cultured in IMEM supplemented with 10% FBS. The human prostate cancer cell lines DU145 and PC3 and the human hematological cancer cell lines Raji, Namalwa, and Ramos (all from ATCC) were cultured in RPMI-1640 medium supplemented with 10% FBS. Caco-2 human enterocytes, Heb3B human hepatoma, and ACHN human renal adenocarcinoma cell lines (all from ATCC) were cultured in DMEM supplemented with 10% FBS. All cells were incubated at 37°C in a humidified atmosphere with 5% CO2.
Rhegf
Manufactured by Promega
Sourced in United States
RhEGF is a recombinant human epidermal growth factor (EGF) product manufactured by Promega. It is a protein that promotes cell growth and proliferation in various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
293t transfection reagent, by Mirus Bio (2 mentions)
Activin a, by Merck Group (2 mentions)
Rhfsh, by Merck Group (2 mentions)
Rhfsh, by Promega (2 mentions)
α mem, by Thermo Fisher Scientific (2 mentions)
Recombinant human insulin rh insulin, by Thermo Fisher Scientific (1 mentions)
Rwpe 1, by Thermo Fisher Scientific (1 mentions)
Rhegf, by Thermo Fisher Scientific (1 mentions)
Keratinocyte serum free medium, by Thermo Fisher Scientific (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Selenium, by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
Transferrin, by Merck Group (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Transferrin, by Thermo Fisher Scientific (1 mentions)
Selenium, by Thermo Fisher Scientific (1 mentions)
Insulin, by Thermo Fisher Scientific (1 mentions)
Ksom aa, by Merck Group (1 mentions)
Sm22α, by Abcam (1 mentions)
9 protocols using rhegf
1
Cell Culture Conditions for Diverse Cell Lines
2
Genetically-Engineered Cell Lines for Oncology Research
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K-Ras mutant lung and pancreatic carcinoma lines LKR10 (mouse lung), A549 (human lung) and CFPac1 (human pancreas) were used (all these cell lines are wild-type for EGFR, see below and the COSMIC database for the human cell lines). 293T, LKR10, A549 and CFPac1 cells were grown in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 10% fetal calf serum (FCS, GIBCO), 100 units/ml penicillin/streptomycin and glutamine. All cells were cultured at 37°C in a humidified incubator with 5% CO2. For transient expression, cells were transfected using Mirus 293T transfection reagent and collected 24 to 36 hours later. For stable knockdown, cells were transduced with lentiviral shRNA constructs using the packaging vectors pGagpol and pΔ8.2, followed by 2 μg/ml puromycin selection for one week. For rescue experiments, cells were transduced with retroviral pBabe and pMSCV constructs using packaging the pGag and pVSVg vectors, followed by 100 μg/ml hygromycin selection for one week.
Serum and EGF stimulation were performed after 48h of serum-starvation using either regular 10% FBS cell media or 25 ng/μl of rhEGF (Promega) for 15 min.
Serum and EGF stimulation were performed after 48h of serum-starvation using either regular 10% FBS cell media or 25 ng/μl of rhEGF (Promega) for 15 min.
3
Ovarian Tissue Culture and IVF Protocol
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Ovarian tissue culture was performed using in vitro growth (IVG) medium consisting of αMEM (Invitrogen, CA) supplemented with 5% FBS, 100 mIU/ml rhFSH (MERCK, NJ), 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium (Sigma-Aldrich, MO). Activin A (Sigma-Aldrich, MO) was used as an IVG supplement (30 ng/ml). IVM medium consisted of αMEM supplemented with 5% FBS, 100 mIU/ml rhFSH and 5 ng/ml rhEGF (Promega, WI). TYH medium was used for IVF. After IVF, fertilized oocytes and embryos were cultured in mCZB medium.
4
Culturing Oncosphere Cancer Cells
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After sorting, cells were suspended in 1 mL MEBM (Lonza-Cloetics, Basel, Switzerland) supplemented with B27 (Thermo Fisher Scientific—Gibco, Reinach, Switzerland), 20 ng/mL rhEGF (Promega, Dübendorf, Switzerland), 20 ng/mL bFGF (Gibco, Thermo Fisher Scientific, Reinach, Switzerland), rhInsulin (Amimed, BioConcept, Allschwill, Switzerland), 100 U/mL penicillin and 100 µg/mL streptomycin. Cells were seeded in ultra-low attachment 24-well plates (Corning® Costar, Sigma-Aldrich, Buchs, Switzerland) with a density of 1000 cells/mL. After 14 days, spheres with a diameter of at least 50 nm were manually counted.
5
Ovarian tissue culture for in vitro growth
6
Notoginsenoside R1 Signaling Pathway
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Notoginsenoside R1 (NGR1, chemical structure C47H80O18, molecular weight = 933, purity >98%) was purchased from Chinese National Institute for the Control of Pharmaceutical and Biological Products. rhEGF and rhFGF were purchased from Promega (Madison, WI). The small molecule inhibitors LY294002 (PI3K/Akt inhibitor), SPD98059 (ERK inhibitor), SB203580 (p38 MAPK inhibitor), SP600125 (JNK inhibitor), BrdU were obtained from Sigma-Aldrich (St. Louis, MO). Primary antibodies against Akt/phospho-Akt, ERK/phospho-ERK, p38/ phospho-p38 and JNK/ phospho-JNK were all purchased from Cell Signaling Technology (Massachusetts, USA). Antibodies against Myosin-11, SMA, Calponin, SM22-α and BrdU were obtained from Abcam (Cambridge, UK). See Supplementary Tables 1 -2 for detailed information.
7
Genetically-Engineered Cell Lines for Oncology Research
8
Glioblastoma Stem Cell Isolation
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The GSCs were obtained by dissecting GBM tumor tissue and then treating with trypsin, followed by a trypsin inhibitor. The chopped tissue was then passed through a cell strainer to remove the debris. The obtained filtrate was plated in ultra-low attachment plates using the stem cell for neurosphere formation media described next. Then, neurospheres were grown in Neurobasal medium (#21103049, Gibco) supplemented with 1× l -glutamine (#25030081, stock 200 mM, i.e., 100×, Gibco), heparin 2 μg/ml (#H3393 Sigma), 1× B27 supplement (#17504044, Gibco, stock concentration 50×), 0.5× N2 supplement (#17502048, Gibco, stock concentration 100×), 20 ng/ml rhEGF (#g5021, Promega), 2 ng/ml rhFGF-basic (#100-18B-100 μg, PeproTech), and penicillin and streptomycin. To make single-cell suspensions for re-plating, the spheres were chemically dissociated after 7 days of plating, using NeuroCult Chemical Dissociation Kit (mouse, #05707) from Stem Cell Technologies, according to the manufacturer’s instructions.
9
Competitive EGFR-Nanoparticle Uptake Assay
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To further investigate the competitive effect of EGFR on the uptake of the nanoparticles, tumor cells were seeded in 24-well plates overnight and were incubated with 131 I-EGFR-BSA-PCL and 131 I-BSA-PCL for 1 h; radioactivity was measured using a γ counter (LKB gamma 1261; LKB Instruments). Next, 500 μL rhEGF (0.2 ng/mL, Promega, USA) was added to these cells for 1 h, and then tumor cells were incubated with 131 I-EGFR-BSA-PCL for another 1 h. After incubation, cells were washed three times with PBS before radioactivity was measured.
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