Effect of vorinostat on IGF-I-mediated signal transduction.
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Ishikawa and USPC-2 cells were treated for 24 h with vorinostat (or left untreated) and/or IGF-I during the last 10 min of the incubation period.
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Induction of lamellipodia formation and invasion by IGF-I in MDA-MB-231 cells but not MCF7 cells.
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(a) After stimulation with or without (mock) IGF-I, cells were stained with phalloidin.
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Cell preparation
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Human ESC-CMs and non-cardiac hESC derivatives were harvested and cryopreserved after 16–18 days under differentiating conditions.
- One day prior to harvest, cells were subjected to a pro-survival protocol, previously shown to enhance engraftment post-transplantation.
- In brief, cultures were heat-shocked with a 30-minute exposure to 43 °C medium, followed by RPMI-B27 medium supplemented with IGF-1 (100 ng/ml) and cyclosporine A (0.2 μM, , ).
- One day later, cultures were harvested with 0.25% trypsin/0.5 mM EDTA and cryopreserved as previously described.
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Materials
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Hank's Balanced Sodium Salt (HBSS), medium'>Dulbecco's medium'>modified medium'>Eagle's medium (DMEM) high glucose (4.5 g/L), alpha minimum essential medium, phosphate-buffered salt (PBS), penicillin/streptomycin, trypsin/EDTA (0.05%/0.53 mM), L-glutamine, superscript III kit, NuPAGE 4%–12% Bis–Tris gel, bone morphogenetic protein-2 (BMP-2), and polyvinylidene difluoride (PVDF)membranes were obtained from .
- Low-molecular weight heparin (4,500 g/mol) was obtained from (, http://www.sanofi.com/).
- 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) reagents were from (, http://www.promega.com/products).
- Total collagens and GAG quantification kits were from (, http://www.biocolor.co.uk).
- Brilliant SYBR Green Master Mix was obtained from ( , The http://www.stratagene.com).
- PCR primers were synthesized by MWG (, http://www.mwg-biotech.com).
- TGF-β1 and Insulin-like growth factor-1 (IGF-1) were obtained from (, http://www.peprotechec.com).
- Protein content was determined using the assay (, , http://www.piercenet.com).
- The Western blot detection system was obtained from GE Healthcare (Buckin-ghamshire, UK, http://www3.gehealthcare.com).
- All other chemicals were obtained from standard laboratory suppliers and were of the highest purity…
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Effects of IGF-1-containing gelatin hydrogel on neurogenesis in the SVZ.
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(b) Coronal sections of brains that received an injection of microspheres containing IGF-1 or microspheres plus PBS, showing DCX+ new neurons in the SVZ (green) (n = 5 animals for each group).
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Effects of Activin A on paraxial mesodermal differentiation of mouse iPS cells.
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The cultures also contained BMP4 (10 ng/ml), IGF-1 (10 ng/ml), LiCl (5 mM), and Shh (10 ng/ml).
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In vitro Embryo Cultures
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Tri-pronuclear zygotes were cultured individually in 30 µl microdrops containing the Global-Medium with 5% human serum albumin in the presence or absence of EGF, IGF-I, GM-CSF, BDNF, and CSF-1 , all at 10 ng/ml.
- The culture medium was renewed every 48 h. Embryonic development was evaluated at 96, 120, and 144 h after culture.
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In vitro Embryo Cultures
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Normally fertilized embryos frozen on day 3 of culture by slow cooling were thawed by using a 2-step thawing protocol 2, 5% O2 and 90% N2 with or without growth factor mixtures containing 10 ng/ml of EGF, IGF-I, GM-CSF, BNF, CSF-1, artemin, and GNF .
- Individual embryos were cultured for 72 h in a 30 µl drop of medium and their development was evaluated.
- The doses of these growth factors chosen for these experiments were based on previous studies (BDNF
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Cell cycle analysis of MCF-7 cells in the presence of IGF-1 at 8 h. MCF-7 cells were serum starved for 24 h. At 22 h after serum starvation the cells were treated with 10 μM auraptene in 0.01% DMSO.
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At 24 h serum starvation, the cells were treated with IGF-1 (10 ng/mL).
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Effect of metformin on IGF-I-mediated signal transduction and mTOR and Ampk signalling pathway in endometrial cancer cells.
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A, Ishikawa, ECC-1, USPC-2 and USPC-1 cells were treated with metformin (10 mM) for 24 h (or left untreated) in the presence or absence of IGF-I (50 ng/ml) during the last 10 min of the incubation period.
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