The mouse kidney was digested into single cells as has been described before28 (link). Briefly, the mouse kidney was decapsulated and chopped into small pieces of 2–3 mm of diameter, followed by 45 minutes’ digestion with 40 mg/dl collagenase (Sigma-Aldrich) in a 37 °C shaker at 200 rpm. The digestion appeared to be very complete, since most of the digests passed a 66 nm filter. The filtered kidney digests were then incubated with FITC-conjugated anti-E-cadherin (E-cad, Becton-Dickinson Biosciences, San Jose, CA, USA) for labeling of the renal epithelial cells. FITC-positive cells were sorted by flow cytometry. The sorted cells were either analyzed immediately or cultured in RPMI1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 15% fetal bovine serum (FBS, Sigma-Aldrich, St Louis, MO, USA), penicillin (100 μg/ml) and streptomycin (250 ng/ml) in a humidified chamber with 5% CO2 at 37 °C. Recombinant human IGF1 (100 ng/ml) and ERK/MAPK-p42/p44 inhibitor PD98059 (10 μmol/l) were also purchased from Sigma-Aldrich.
Recombinant human igf 1
Manufactured by Merck Group
Sourced in United States
Recombinant human IGF-1 is a laboratory product produced using recombinant DNA technology. It is a protein that is structurally and functionally identical to the naturally occurring insulin-like growth factor 1 found in humans.
Automatically generated - may contain errors
Lab products found in correlation
Igf 1, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Ly294002, by Merck Group (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Collagenase, by Merck Group (1 mentions)
Pd98059, by Merck Group (1 mentions)
Low attachment six well plates, by Corning (1 mentions)
Biostation ct, by Nikon (1 mentions)
Lps escherichia coli serotype 026 b6, by Merck Group (1 mentions)
Effectene transfection reagent, by Qiagen (1 mentions)
Dual glo luciferase assay, by Promega (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Igf 1, by R&D Systems (1 mentions)
3 3 5 triiodo l thyronine t3, by Merck Group (1 mentions)
Hrp conjugated anti rabbit antibody, by Santa Cruz Biotechnology (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Streptavidin alexa fluor 594 conjugate, by Thermo Fisher Scientific (1 mentions)
Anti erk, by Santa Cruz Biotechnology (1 mentions)
25 protocols using recombinant human igf 1
1
Isolation and Culture of Mouse Kidney Epithelial Cells
2
Differentiation of hESCs into Ventral Neural Lineages
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hESC (line H9, WiCell) was expanded and differentiated as previously described 21 ; however, EBs were not transferred to attachment culture conditions as done previously, but were maintained in 3D culture throughout differentiation using bacteriological dishes and low attachment six‐well plates (Corning, New York, USA). For IGF‐1 treatment, cultures were differentiated in ventral neural induction media (VNIM) media 21 supplemented with recombinant human IGF‐1 (5 ng/ml, Sigma‐Aldrich, San Louis, USA) until day 37, then in basal knockout serum‐free media 14 with 10 ng/ml IGF‐1 until day 90 (Fig. 1 Aii). Control cultures were differentiated in parallel in the absence of IGF‐1 (Fig. 1 Ai). Time‐lapse capture of culture morphology was achieved using a BioStation CT (Nikon Corporation, Tokyo, Japan).
3
Luciferase Assay for Innate Immune Response
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Defensin and Gambicin promoters were cloned into the promoter-less luciferase reporter pGL3 (Promega; Madison, Wisconsin). Transfections of A. stephensi ASE cells were performed using Effectene transfection reagent (Qiagen; Valencia, CA) as previously described [14] (link). At 24 h post-transfection, cells were challenged with 100 µg/ml LPS (Escherichia coli serotype 026:B6; Sigma-Aldrich) with or without 0.013–1.3 µM recombinant human IGF1 (Sigma-Aldrich). Luciferase activity was measured by microplate reader (Hidex Chameleon, LabLogic; Brandon, FL) using the Dual-Glo Luciferase Assay (Promega) at 24 h post-immune challenge. Data were analyzed by unpaired t-tests comparing each treatment to matched buffer controls. Experiments were replicated three (Gambicin) or four (Defensin) times.
4
Serum-starvation and IGF-1 Stimulation
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Prior to IGF-1 stimulation experiments (long-term degradation or short-term signalling), attached cells were washed with PBS and changed to serum-free medium and incubated at 37 °C for 8–12 h. Stimulation was preformed using recombinant human IGF-1 (Sigma-Aldrich Ltd., St Louis, MO, USA) at 50 ng/ml.
5
Oligodendrocyte Progenitor Cell Generation
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NG2+/Olig2+ progenitors were generated from the NSCs by culturing in OPC medium [DMEM/F12 mixture medium (Sigma-Aldrich, United Kingdom), 1% N2 supplement, 0.5% B-27 supplement, 1% GlutaMAX, 1% Penicillin-Streptomycin, 20 ng/ml FGF-2, 100 ng/ml recombinant human insulin like growth factor-1 (IGF-1, R&D systems), and 20 ng/ml recombinant human platelet-derived growth factor α chain, homodimer (PDGF-AA, Generon Ltd., Maidenhead, United Kingdom)]. Medium change was performed in every other day. After reaching sub-confluency, the cultured cells were split into new culture dishes. In this study, we defined the cultured cells as NOP after maintaining under this condition for at least 7 days. To promote further oligodendrogenesis, NOP were plated in culture dishes filled with OPC medium. After growing at confluency, culture media was changed to OL medium [DMEM/F12 mixture medium, 1% N2 supplement, 2% B-27 supplement, 1% GlutaMAX, 1% Penicillin-Streptomycin, 100 ng/ml recombinant human IGF-1, 40 ng/ml 3,3′,5-Triiodo-L -thyronine (T3, Sigma-Aldrich), and 3.5 ng/ml Hydrocortisone (Sigma-Aldrich)], and the cells were maintained for 6 days to promote terminal differentiation into the OL lineage (NOP/OL). The cell culture protocols for generating NOP and NOP/OL are illustrated in Supplementary Figure 1 .
6
Embryokine Treatment of Zygotes
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The procedure for treatment of embryos consisted of replacing 5 μl culture medium in the microdrop with 5 μl SOF-BE2 containing the embryokine at ten times the final concentration or the relevant vehicle. The final concentrations of embryokine tested were 100 ng/ml for IGF1, 1 nM for activin A and 66 ng/ml for WNT7A. Treatments concentrations were chosen because they were effective at increasing the proportion of putative zygotes becoming blastocyst [13 , 16 (link), 17 (link)]. Human activin A (amino acid sequence identity with bovine βA inhibin = 95%) and recombinant human IGF1 (amino acid sequence identity with bovine IGF1 = 95%) were obtained from Sigma-Aldrich, whereas recombinant human WNT7A (amino acid sequence identity with bovine WNT7A = 99%) was purchased from eBioscience Inc. (San Diego, CA, USA). The vehicle was SOF-BE2 diluted 1:5 (v/v) with water for IGF1, Dulbecco’s phosphate-buffered saline (DPBS) with 0.1% (w/v) bovine serum albumin (BSA) for activin A and a mixture of 97% SOF-BE2 (v/v) and 3% (v/v) of 10 mM NaPO4, 500 mM NaCl and 0.5% (w/v) CHAPS diluted 1:100 (v/v) in DBPS-BSA for WNT7A.
7
Cellular Signaling Pathway Modulation
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Glucosamine (GlcN), Thiamet G (TG), Thiazolyl Blue Tetrazolium Bromide (MTT), Cisplatin, pNPP (4-Nitrophenyl phosphate di(2-amino-2-ethyl-1, 3-propanediol)), and recombinant human IGF-1 were from Sigma-Aldrich. Anti-phospho-Akt, anti-phospho-Erk, anti-IGF-1R and anti-PTP1B antibodies were from Cell Signaling Technology. Anti-Akt, anti-Erk, anti-GAPDH, and HRP-conjugated anti-rabbit antibodies were from Santa Cruz Biotechnology. Anti phospho-IGF-1R antibody (anti-phospho-IR/IGF1R pTyr1158/1162–1163) was from Sigma-Aldrich. Immunoprecipitation of PTP1B for enzymatic assays was performed using anti-PTP1B antibody from Calbiochem. Anti-O-GlcNAc antibody (RL2) and Streptavidin Alexa Fluor 594 conjugate were from Thermo Fisher Scientific. HRP-conjugated anti-mouse, Fluorescein (FITC)-AffiniPure Goat anti-Mouse, and Biotin-SP-Conjugated AffiniPure Goat anti-Rabbit were from Jackson ImmunoResearch Laboratories.
8
Prostate Cancer Cell Adhesion Assay
9
Gastric Cancer Cell Line Culturing and Antibody Analysis
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GES-1, an immortalized human gastric epithelial cell line, was also purchased from ATCC, and cells (passages 5–10) were maintained in Gibco RPMI-1640 (Thermo Fisher Scientific, Inc.) medium supplemented with 10% FBS, 2 mM L-glutamine (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin. The human gastric cancer cell lineMGC803, BGC823and HGC27 were obtained from American Type Culture Collection (ATCC, Manassas, Va.), and was cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Hyclone) and 100 U/mL penicillin/streptomycin (Gibco) and were maintained in a humidified atmosphere with 5% CO2 at 37°C. All experiments were performed in the absence of FBS. Recombinant human IGF-1 was obtained from Sigma Corporation (St. Louis, USA). All antibodies in this experiment can be purchased from Santa Cruz Biotechnology (Santa Cruz, USA), involving anti-p-AKT, anti-t-AKT, anti-SRPK1 (Santa Cruz Biotechnology Cat. number sc-289; 1:200); anti-N-cadherin,anti-MMP2,anti-Slug, and anti-β-actin. All assays were repeated three times.
10
Fibroblast Isolation and IGF-1 Treatment
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The isolation of fibroblasts from young (3-6 mos) and aged (21-24 mos) wild-type and Ames dwarf mice has been described [23 (link)]. In this study, the fibroblasts at passage 4 or 5 were plated in 100 mm2 cell culture dishes (2×105 cells/dish) and cultured for 3 days in DMEM medium containing 15% FBS. The day before treatment, the medium was replaced with a medium containing 0.5% FBS. The recombinant human IGF-1 (Sigma) was prepared in distilled H2O and filter sterilized. The IGF-1 stock solution (25 ng/ml) was diluted in DMEM medium containing 0.5% FBS and the fibroblasts were treated with IGF-1 at a final concentration of 2 ng/ml. The cells were harvested and cytoplasmic and nuclear extracts were prepared [23 (link)]. The protease and phosphatase inhibitors were added to the extraction buffers prior to use [23 (link)]. The protein concentration of the extracts was determined using Bradford reagent (Bio-Rad).
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