The human thyroid cell line KAT18, human papilloma thyroid cancer cell line TPC-1, human follicular thyroid cancer cell line FTC-133 derived from lymph node metastasis of follicular thyroid cancer, human thyroid cancer papillary cell line BCPAP, and Nthy-ori 3–1 control cells were from ProCell (Wuhan, China), and were grown in RPMI-1640 (Gibco, MA, USA) with 10% FBS (Gibco), streptomycin (100U/mL), and penicillin sodium (100U/mL). The culture conditions were 37 °C and 5% CO2. IGF-1) treatment, cells were grown in media containing 400 ng/mL IGF-1 (Sigma-Aldrich, MO, USA) at 37 °C for 24 h. Following IGF-1 treatment, the milieu was substituted with fresh milieus. Cells were tested for mycoplasma contamination approximately once a month using the MycoAlert Mycoplasma Detection Kit (Cat. No. LT07-218; Lonza Cologne GmBH, Cologne, Germany).
Igf 1
Manufactured by Merck Group
Sourced in United States, Germany, Switzerland, Italy, Japan, Spain, Sao Tome and Principe, United Kingdom
IGF-1 is a laboratory assay kit used to measure the levels of insulin-like growth factor 1 (IGF-1) in biological samples. IGF-1 is a hormone that plays a crucial role in growth and development processes.
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Lab products found in correlation
Epidermal growth factor (egf), by Merck Group (21 mentions)
Insulin, by Merck Group (15 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (12 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (10 mentions)
Heparin sulfate, by Merck Group (9 mentions)
Ly294002, by Merck Group (9 mentions)
Igf 1, by Thermo Fisher Scientific (8 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (8 mentions)
Dmem f12, by Thermo Fisher Scientific (8 mentions)
Igf 1, by R&D Systems (6 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (6 mentions)
Activin a, by Thermo Fisher Scientific (6 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (6 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (6 mentions)
Glutamax, by Thermo Fisher Scientific (5 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Dexamethasone (dex), by Merck Group (5 mentions)
Bovine serum albumin (bsa), by Merck Group (5 mentions)
L glutamine, by Thermo Fisher Scientific (5 mentions)
175 protocols using igf 1
1
Thyroid Cancer Cell Line Characterization
2
Time-dependent Effects of cGP and IGF-1 on Cell Viability
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A total cell count assay was used to determine time-depended effects of cGP and/or IGF-1 under conditions of either serum enrichment (0.2% FBS, Fig. 2a & b ) or serum withdraw (Fig. 2c ). The assay enables an evaluation of temporal changes over a 6 day period at multiple time points. The total number of cells was determined by seeding cells in 6-well plates at a density of 15,000 cells/well and the assays were set up in triplicate. After overnight incubation in complete medium, cells were washed twice with PBS and compounds in 0.2% serum containing media were added to the wells. The compounds used were IGF-1 alone (100 nM, Genentech, San Francisco, CA, USA), cGP alone (100 nM, Bachem AG, Switzerland), IGF-1 + cGP (100 nM), GPE alone (100 nM, Sigma), GPE + IGF-1 (100 nM) and IGF-1 + cGP + GPE (100 nM). Media and compounds were replaced every 48 h. Cells were trypsinised and counted every 24 h from day 4 to day 6. Cell counting was done using a hematocytomter. Ten μl of cell suspension was pipetted onto each grid and all 9 squares were counted. Eight counts were performed for each well and cells/ml was calculated using the formula: cells/ml = (Average of counts/9) × 10,000.
3
IGF-1 Signaling Pathway Modulation
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U2-OS, MCF7, IMR90, HCT116, A549, and MEF cells were maintained in DMEM supplemented with 10% (12% for MEFs) FBS and 1% penicillin/streptomycin sulfate (Invitrogen Inc., Carlsbad, CA, USA) at 37 °C in a humidified incubator under 5% CO2. Prior to IGF-1 treatment, MCF7 and HCT116 cells at 60–70% confluency were incubated in serum-free DMEM for 48 h, while MEFs and IMR90 cells were serum-starved in DMEM containing 0.5% FBS. IGF-1 (human recombinant; Sigma, St. Louis, MO, USA) was prepared as a 100 μg mL−1 stock solution in PBS according to manufacturer’s instructions. LY294002 (Calbiochem, San Diego, CA, USA) was prepared as a 25 mm stock solution. Trichostatin A (TSA; Sigma) was prepared as a 2 mg mL−1 stock in DMSO. At 45 min prior to IGF-1 treatment, LY294002 was added to serum-starved cells at a final concentration of 25 μm . For acetylation of endogenous p53, cells were deprived of serum for 48 h before treatment with IGF-1 for 12 h and subsequently treated with 40 μm Trichostatin A for 6 h. Nutlin-3a (Sigma) was used at 5.0 nm either with IGF-1 or with the vehicle control.
4
FOXO1 Expression in MC3T3-E1 Cells
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MC3T3-E1 cells (10,000 cells/well) were seeded on a 12-well plate and subjected to different treatments (Con, Dex and Dex+IGF-1) for 48 h. In Dex+IGF-1 group, MC3T3-E1 cells were pre-incubated with IGF-1(50 ng/mL; Sigma, USA) for 2 h, and then co-incubated with 200 µM Dex for 48 h. After washed by PBS for three times, cells were fixed with 4% formaldehyde and permeabilized by the assistant of 0.5% Triton X-100. Subsequently, cells were blocked with 5% bovine serum albumin (BSA), co-incubated with the primary antibody (FOXO1) overnight at 4 °C and FITC-conjugated goat secondary antibody the next day. The nuclei were stained with DAPI, and the outcomes were acquired using a fluorescence microscope.
5
Adipogenic Differentiation of Rat Tendon Stem Cells
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All experiments were approved by the Animal Research Ethics Committee, Third Military Medical University, China. Rat TSCs were isolated from Sprague-Dawley rats and cultured, as described previously [21] (link). To determine if PGE2 induced adipogenic differentiation, TSCs were seeded in six-well plates at a density of 6×104/well and were incubated with 0, 10, 50, 100 or 200 ng/ml PGE2 (Sigma-Aldrich, St. Louis, MO, USA) for 7 days, or with PGE2 100 ng/ml for 0, 3, 7 or 10 days. To determine the ability of BMP-2 to induce adipogenic differentiation, rat TSCs were incubated with 0, 10, 50, 100 or 200 ng/ml BMP-2 (Sigma-Aldrich) for 7 days, or with BMP-2 (100 ng/ml) for 0, 3, 7 or 10 days. The effects of IGF-1 on adipogenic differentiation of rat TSCs were determined by incubation with IGF-1 (Sigma-Aldrich) alone (10 nM), or with 0, 1, 5, 10 or 20 nM IGF-1 in combination with 100 ng/ml BMP-2. The cAMP synthesis inhibitor 2′,5′-dideoxyadenosine (ddA, 10 μmol/l, Sigma-Aldrich) and the PKA inhibitor H-89 (10 μM, Sigma-Aldrich) were added to PGE2-stimulated TSCs to determine the role of the cAMP/PKA/CEBPδ pathway.
6
IGF1 Enrichment of Colon Cancer Cells
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The human colon cancer cell lines HCT116 and DLD-1 used in this study were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and maintained and passaged according to the protocols provided. For CSC-enrichment experiments, IGF1 (200 ηg/mL, Millipore, Taipei, Taiwan) was added to the culture medium for 48 h (after the first 24 h, the culture medium was discarded and fresh medium containing IGF1 was replenished and cells cultured for an additional 24 h). IGF1-treated cells were then harvested for further analyses.
7
Cellular Signaling Modulation Assays
8
Fetal Islet Proliferation Assay
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Islets from three fetuses were used for each hormone study. At least 50 islets in each Petri dish were incubated with either T3 (Sigma), insulin (Humulin‐R, Lilly, Indianapolis, IN, USA) or leptin (Abcam, Cambridge, UK) at three doses: 0.1, 1 and 10 ng ml−1. The effects of T4 were not assessed as T3 is the active form of thyroid hormone. An additional dish of islets incubated with 10 ng ml−1 IGF‐I (Sigma) was used as a positive control as IGF‐I is known to stimulate beta cell proliferation (Hogg et al. 1993 ). The islets were subjected to 95% O2/5% CO2 gas for 5 min in a humidity chamber before incubation at 37°C. After 24 h, the islets were transferred into fresh medium supplemented with hormone and 5‐ethynyl‐2′‐deoxyuride (EdU) at a final concentration of 10 μm . After a total incubation time of 48 h, the islets were fixed in 4% paraformaldehyde and frozen in optimum cutting temperature compound (Tissue Tek, Torrance, CA, USA). Islets were sectioned at −20°C to a thickness of 10 μm at 100 μm intervals and labelled for EdU‐positive cells using the Click‐iT EdU Alexa Fluor‐488 kit (Invitrogen, Grand Island, NY, USA).
9
Plasma IGF-1 and Phenylalanine Enrichment
10
Epigenetic and Growth Signaling Modulation
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Cells were seeded and treated with 5-Aza-dC (5 μM; Sigma). After incubating for 96 hours with a change of culture medium every 24 hours, cells were collected for further analysis.
Cells were seeded overnight and refreshed with serum-free medium, then subjected to serum starvation for 12 hours. After starvation, cells were treated with vehicle, 10% fetal bovine serum (FBS) or 10 nM IGF1 (Sigma) for different durations. For experiments involving inhibition of signaling pathways, cells were cultured in the presence of PD98059 (Sigma) or LY294002 (Sigma), specific inhibitors of MAPK kinase and PI3K, respectively, for 1 hour prior to IGF1 stimulation.
Cells were seeded overnight and refreshed with serum-free medium, then subjected to serum starvation for 12 hours. After starvation, cells were treated with vehicle, 10% fetal bovine serum (FBS) or 10 nM IGF1 (Sigma) for different durations. For experiments involving inhibition of signaling pathways, cells were cultured in the presence of PD98059 (Sigma) or LY294002 (Sigma), specific inhibitors of MAPK kinase and PI3K, respectively, for 1 hour prior to IGF1 stimulation.
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