Insulin like growth factor 1 igf 1

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Insulin-like growth factor-1 (IGF-1) is a laboratory reagent used in various research and diagnostic applications. It is a polypeptide hormone that plays a key role in cell growth, development, and metabolism. IGF-1 functions by binding to specific receptors on target cells, triggering signaling cascades that regulate cellular processes.

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33 protocols using insulin like growth factor 1 igf 1

Ovarian cell culture protocol

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Medium 199, McCoy’s 5A medium, and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). Percoll, insulin-transferrin-selenium mix, and deoxyribonuclease (DNase) I were obtained from Sigma-Aldrich (St. Louis, MO). Collagenase type 1 was purchased from Worthington (Lakewood, NJ), and insulin-like growth factor–I (IGF-I) was provided by PeproTech (Rocky Hill, NJ). Estradiol valerate was obtained from Bayer (Progynova; Bayer, Leverkusen, Germany), and progesterone was purchased from Pfizer (Provera; Pfizer Inc., NY, USA). Primary antibodies for PCNA and cyclin D1 were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against FSH-R, LH-R, CYP17A1, and CYP19 were obtained from Abcam (Cambridge, UK). β-Actin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Sex steroid (estrogen and progesterone) enzyme-linked immunosorbent assay (ELISA) kits were provided by Enzo, Life Sciences (Plymouth Meeting, PA, USA). The FSH and LH ELISA kits were purchased from Tsz Biosciences (San Francisco, CA, USA). The vendors for other chemicals and reagents are denoted in the corresponding sections of Methods.

Yoon H.J., Lee Y.J., Baek S., Chung Y.S., Kim D.H., Lee J.H., Shin Y.C., Shin Y.M., Ryu C., Kim H.S., Ahn S.H., Kim H., Won Y.B., Lee I., Jeon M.J., Cho S.H., Lee B.S., Sung H.J, & Choi Y.S. (2021). Hormone autocrination by vascularized hydrogel delivery of ovary spheroids to rescue ovarian dysfunctions. Science Advances, 7(18), eabe8873.

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Isolation and Culturing of Pancreatic Islet Cells

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Medium 199 and McCoy’s 5 A media were purchased from Gibco-BRL (Life Technologies/Gibco-BRL, Grand Island, NY). Percoll, FSH, LH, insulin-transferrin-selenium mix (ITS), deoxyribonuclease I (DNase 1), poly-L-ornithine (PLO molecular weight 15–30 KDa) were purchased from Sigma-Aldrich (St. Louis, MO). Low viscosity (20–200 mPa·s) ultra-pure sodium alginate with high mannuronic acid (LVM) content and with high guluronic acid (LVG) were purchased from Nova-Matrix (Sandvika, Norway). Collagenase type 1 was from Worthington (Lakewood, NJ) and insulin-like growth factor-I (IGF-I) from Peprotech (Rocky Hill, NJ). Solutions for alginate microcapsule synthesis were made using the following chemicals: HEPES, sodium chloride, calcium chloride, and strontium chloride (Fisher Scientific, Pittsburgh, PA). The vendors for other chemicals, reagents, assay kits and antibodies used have been indicated in the relevant areas of this method section.

Sittadjody S., Saul J.M., McQuilling J.P., Joo S., Register T.C., Yoo J.J., Atala A, & Opara E.C. (2017). In vivo transplantation of 3D encapsulated ovarian constructs in rats corrects abnormalities of ovarian failure. Nature Communications, 8, 1858.

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Tumor Cell Signaling Pathway Modulation

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Tumor cells were cultured for 24 hours with the following murine factors: 2 ng/mL TGF-β1 (R&D Systems), 2 ng/mL TGF-β2 (R&D Systems), 50 ng/mL Sonic Hedgehog (Shh) (PeproTech, #315-22), 50 ng/mL WNT-3A (PeproTech, #315-20), 100 ng/mL Insulin-Like Growth Factor 1 (IGF-1) (PeproTech, #250-19), 50 ng/mL Epidermal growth factor (EGF) (PeproTech, #315-09), 50 ng/mL Fibroblast Growth Factor 2 (FGF2 or bFGF) (PeproTech, #450-33), 100 ng/mL Osteopontin (OPN) (Leinco, #O121), 10 ng/mL Interleukin 4 (IL-4) (R&D systems), 200 ng/mL Stromal Cell-derived Factor 1α (SDF-1α or CXCL12) (Biolegend), 10 ng/mL Interleukin 6 (IL-6) (R&D systems).

Ross M.H., Esser A.K., Fox G.C., Schmieder A.H., Yang X., Hu G., Pan D., Su X., Xu Y., Novack D.V., Walsh T., Colditz G.A., Lukaszewicz G.H., Cordell E., Novack J., Fitzpatrick J.A., Waning D.L., Mohammad K.S., Guise T.A., Lanza G.M, & Weilbaecher K.N. (2017). Bone-induced expression of integrin β3 enables targeted nanotherapy of breast cancer metastases. Cancer research, 77(22), 6299-6312.

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Investigating Molecular Pathways in Cancer

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Luteolin and phalloidin were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Insulin-like growth factor-1 (IGF-1) was purchased from PeproTech, Inc. (Rocky Hill, NJ, USA) Anti-phosphorylated (p-)insulin-like growth factor-1 receptor (IGF-1R) (cat. no. sc-81499; 1:500) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-matrix metalloproteinase (MMP)-2 (cat. no. ab-7033; 1:1,000), anti-MMP-9 (cat. no. ab-76003; 1:1,000), anti-tissue inhibitor of metalloproteinase (TIMP)-1 (cat. no. ab-109125; 1:1,000) and anti-TIMP-2 (cat. no. ab-157386; 1:1,000) were purchased from Abcam (Cambridge, UK). Anti-E-cadherin, anti-N-cadherin, anti-vimentin, anti-β-catenin, anti-vimentin (EMT kit; cat. no. cst-9782; 1:1,000), anti-p-protein kinase B (AKT) (cst-4060; 1:1,000), anti-AKT (cat. no. cst-9272; 1:1,000), anti-p-mammalian target of rapamycin (mTOR) (cat. no. cst-2971; 1:1,000), anti-mTOR (cat. no. cst-2983; 1:1,000), anti-β-actin (cat. no. cst-4970; 1:1,000), and horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG; heavy and light chain; cat. no. cst-7074; 1:5,000) secondary antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The goat anti-mouse IgG-HRP were purchased from BioworldTechnology (cat. no. BS12478; 1:10,000; St. Louis Park, MN, USA).

Wang Q., Wang H., Jia Y., Ding H., Zhang L, & Pan H. (2017). Luteolin reduces migration of human glioblastoma cell lines via inhibition of the p-IGF-1R/PI3K/AKT/mTOR signaling pathway. Oncology Letters, 14(3), 3545-3551.

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Isolation and Characterization of EPCs

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Human CD34-positive cells were prepared from the peripheral blood of healthy volunteers as well as coronary artery disease patients. Approximately 2 × 10³cells were cultured in methylcellulose (H4236; StemCell Technologies, Vancouver, BC, Canada) with 100 ng/mL stem cell factor (SCF), 50 ng/mL vascular endothelial growth factor (VEGF), 20 ng/mL interleukin (IL)-3, 50 ng/mL basic fibroblast growth factor (bFGF), 50 ng/mL epidermal growth factor (EGF), 50 ng/mL insulin-like growth factor-1 (IGF-1; all from PeproTech, Rocky Hill, NJ, USA), and 2 U/mL heparin (Wako Pure Chemical Industries, Osaka, Japan) in 3.5-cm dishes. After 21 days, colony formation was observed. We counted the number of total colonies and colonies for small- or large-type EPCs in dishes using a phase-contrast microscope (CK30; Olympus, Tokyo, Japan). Small- and large-type EPCs were identified by visual inspection with an inverted microscope under 40 × magnification. Small-type EPCs were composed of round adhesive cells, and large-type EPCs were composed of spindle-shaped cells [17 (link), 18 (link)].

Sohma R., Sakuma M., Obi S., Nishino S., Inoue K.I., Kishimoto S., Lu T., Toyoda S, & Inoue T. (2023). Effects of the factor Xa inhibitor rivaroxaban on the differentiation of endothelial progenitor cells. BMC Cardiovascular Disorders, 23, 282.

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Akt and PDK1 Activation Assay

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Inactive Akt1, active Akt, active PDK1 and ATP/Mg²⁺ cocktail were purchased from Upstate Cell Signaling Solutions. Mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2) was purchased from Calbiochem. 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phospho-L-serine (18:0,22:6-PS), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (18:0, 22:6-PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (16:0, 18:1-PC), and 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoinositol-3,4,5-trisphosphate (18:0, 20:4-PIP₃) were purchased from Avanti Polar Lipids. Sequencing grade modified trypsin was purchased from Promega. Anti-phospho-Akt (T308) (Cat#: 2965), anti-phospho-Akt (S473) (Cat#: 9271), anti-Akt (Cat#: 9272), anti-protein kinase C zeta (PKCζ)(Cat#: 9368), anti-phospho-PKCζ(T410) (Cat#: 9378), anti-phospho-PDK1(S241) (Cat#: 3061), anti-PDK1 (Cat#: 3062), and anti-GAPDH (Cat#: 2118) antibodies were purchased from Cell Signaling Technology. Anti-phospho-Akt (T34) (Cat#: 23509) was obtained from Abcam. A dilution of 1:1000 was used in western blotting for all of these antibodies. Insulin-like growth factor-1 (IGF-1) was purchased from PeproTech. PBS without Ca²⁺ was purchased from Quality Biological, Inc. Other reagents were purchased from Sigma or Quality Biological, Inc.

Huang B.X., Lee R., Akbar M, & Kim H.Y. (2015). Threonine 34 phosphorylation by phosphoinositide-dependent protein kinase 1 facilitates dissociation of Akt from the plasma membrane. The international journal of biochemistry & cell biology, 64, 195-201.

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Chondrocyte Differentiation of UC-MSCs

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Chondrogenesis induction was conducted following our previous study (Li et al., 2016 (link)). In brief, UC-MSC was induced to chondrocyte-like cells by chondrocytes specific medium. In monolayer culture, UC-MSCs were supplemented with 0.1 mM dexamethasone, 40 mg/ml L-proline, 10 μg/L transforming growth factor beta-1 (TGF-β1, Peprotech, USA), 10 μg/L insulin-like growth factor-1 (IGF-1) (Peprotech, USA), and 1% insulin transferrin selenium (ITS, Invitrogen). The cells were incubated for 3 weeks at 37°C in a humidified atmosphere of 5% CO₂ and the medium changed every 3 days.

Hou W., Duan L., Huang C., Li X., Xu X., Qin P., Hong N., Wang D, & Jin W. (2021). Cross-Tissue Characterization of Heterogeneities of Mesenchymal Stem Cells and Their Differentiation Potentials. Frontiers in Cell and Developmental Biology, 9, 781021.

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Autophagy Modulation in Glioblastoma Therapy

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PP7 was purchased from Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China), and the purity of PPI was ≥98%. N-acetyl-L-cysteine (NAC) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Insulin-like growth factor 1 (IGF-1) and epidermal growth factor (EGF) were purchased from PeproTech (Rocky Hill, USA). 3-methyladenine (3-MA, S2767) and bafilomycin A1 (Baf-A1, S1413) were from Selleck Chemicals (Houston, TX, USA). TMZ was supplied by Tasly Pharmaceutical Co., Ltd. (Tianjin, China). The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologie (Kumamoto, Japan). Hoechst33342/PI kit, cell mitochondrial isolation kit, and dihydroethidium were supplied by Beyotime Institute of Biotechnology (Haimen, China).

Pang D., Li C., Yang C., Zou Y., Feng B., Li L., Liu W., Geng Y., Luo Q., Chen Z, & Huang C. (2019). Polyphyllin VII Promotes Apoptosis and Autophagic Cell Death via ROS-Inhibited AKT Activity, and Sensitizes Glioma Cells to Temozolomide. Oxidative Medicine and Cellular Longevity, 2019, 1805635.

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Maturation Medium for Neural Differentiation

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MN maturation medium contained basal medium that was a 1:1 mixture of N2 and B-27 medium. The B-27 medium contained neurobasal media (GIBCO), 2% B-27 supplement (GIBCO), and 2 mM Glutamax. The following chemicals were added to basal medium freshly before each medium change: 10 ng mL-1 brain-derived neurotrophic factor (BDNF; R&D systems), 10 ng mL-1 insulin-like growth factor 1 (IGF-1; Peprotech), 1 μM cyclic adenosine monophosphate (cAMP; Sigma), 0.2 μg mL-1 ascorbic acid (Sigma), 0.1 μM RA, and 1 μM Pur.

Chen W., Han S., Qian W., Weng S., Yang H., Sun Y., Villa-Diaz L.G., Krebsbach P.H, & Fu J. (2018). Nanotopography Regulates Motor Neuron Differentiation of Human Pluripotent Stem Cells. Nanoscale, 10(7), 3556-3565.

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Coculture of hACs and hUCB-MSCs

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hACs (P2) and hUCB-MSCs (P3) were used. For coculture with direct cell-cell contact, hACs and hUCB-MSCs were mixed directly at ratio of 1 : 1, 3 : 1, and 5 : 1 (hUCB-MSCs : hACs) [7 (link), 9 (link), 29 (link), 30 (link)] and then cultured in basal medium (DMEM-F12, 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin). For indirect coculture, hUCB-MSCs and hACs were cultured with supernatants from each other [31 (link)]. For growth factor induction in monolayer culture, hUCB-MSCs were maintained in basal medium supplemented with 0.1 mM dexamethasone, 40 mg/mL L-proline, 10 μg/L transforming growth factor beta-1 (TGF-β1, Peprotech, USA), 10 μg/L insulin-like growth factor-1 (IGF-1) (Peprotech, USA), and 1% insulin transferrin selenium (ITS, Invitrogen) [32 (link)–35 (link)]. hACs and hUCB-MSCs cultured alone with basal medium were used as controls. All cells were incubated for three weeks at 37°C in a humidified atmosphere of 5% CO₂ and the medium changed every three days. The designated groups for this study were listed in Table 1.

Li X., Duan L., Liang Y., Zhu W., Xiong J, & Wang D. (2016). Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Contribute to Chondrogenesis in Coculture with Chondrocytes. BioMed Research International, 2016, 3827057.

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