Recombinant human tgf β1

Manufactured by Miltenyi Biotec

Sourced in Germany

Recombinant human TGF-β1 is a protein produced through recombinant DNA technology. TGF-β1 is a member of the transforming growth factor beta family and plays a role in various cellular processes.

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7 protocols using recombinant human tgf β1

96-well Library Compound Screening

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The 96-well plate format compound library with NPs, pre-dissolved in dimethyl sulfoxide (DMSO, 10 mM), was obtained from Selleck Chemicals Llc (Houston, Texas, United States). Celastrol and amphotericin B were purchased from Biomol GmbH (Hamburg, Germany) and Sigma-Aldrich (St. Louis, Missouri, United States), respectively, and dissolved in DMSO to gain a 10 mM stock solution. Recombinant human TGF-β1 was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). The miRNAs, transfection reagents and media were obtained from Thermo Fisher Scientific (Waltham, Massachusetts, United States).

Ly T.D., Kleine A., Fischer B., Schmidt V., Hendig D., Kuhn J., Knabbe C, & Faust I. (2020). Identification of Putative Non-Substrate-Based XT-I Inhibitors by Natural Product Library Screening. Biomolecules, 10(10), 1467.

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Inflammatory Cytokine Modulation Assay

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LPS from Escherichia coli O111:B4, PHA from Phaseolus vulgaris, H-89 [protein kinase A (PKA) inhibitor], forskolin (adenylate cyclase activator), and PGE2 were obtained from Sigma-Aldrich. AS-605240 [phosphatidylinositol-3-phosphate kinase (PI3K) inhibitor], PF-04418948 (EP2 receptor antagonist), and L-161,962 (EP4 receptor antagonist) were purchased from Cayman Chemical. Ficoll-Hypaque was obtained from GE Healthcare. Recombinant human TGF-β1, recombinant human M-CSF, recombinant human IL-4, and recombinant human GM-CSF were obtained from Miltenyi Biotech.

Remes Lenicov F., Paletta A.L., Gonzalez Prinz M., Varese A., Pavillet C.E., Lopez Malizia Á., Sabatté J., Geffner J.R, & Ceballos A. (2018). Prostaglandin E2 Antagonizes TGF-β Actions During the Differentiation of Monocytes Into Dendritic Cells. Frontiers in Immunology, 9, 1441.

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Modulation of CD4+ T Cell Differentiation by MSCs

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CD4⁺ T cells were isolated from healthy donors' PBMCs by human CD4 microbeads (Miltenyi Biotec) and resuspended in complete 1640 medium in the presence of anti-CD3/CD28 antibodies (5 μg/mL), anti-IL4 antibody (10 μg/mL) (eBioscience), and anti-IFNγ antibody (10 μg/mL) (eBioscience). MSCs were cocultured with CD4⁺ T cells (1 : 10) in different culturing systems. For Treg induction, recombinant human TGF-β1 (5 ng/mL) (R&D Systems) and IL-2 (5 ng/mL) (Miltenyi Biotec) were added. For Th17 induction, recombinant human TGF-β1 (5 ng/mL), IL-6 (50 ng/mL) (Miltenyi Biotec), and IL-23 (10 ng/mL) (Miltenyi Biotec) were added. In some experiments anti-CCL2 antibody (R&D Systems) was added for neutralization. After coculture for 5 days, floating cells were used to examine Treg and Th17 cell percentages by flow cytometry, and culture supernatant was collected for measuring IL-17A levels by enzyme-linked immunosorbent assay (ELISA) kits (Biolegend). The adherent MSCs were also collected for measuring the gene expression of transforming growth factor- (TGF-) β1, indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), interleukin- (IL-) 6, and chemokine (C-C motif) ligand 2 (CCL2) by real-time PCR.

Sun Y., Deng W., Geng L., Zhang L., Liu R., Chen W., Yao G., Zhang H., Feng X., Gao X, & Sun L. (2015). Mesenchymal Stem Cells from Patients with Rheumatoid Arthritis Display Impaired Function in Inhibiting Th17 Cells. Journal of Immunology Research, 2015, 284215.

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TGF-β1 and SAR405 Protocol

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Recombinant human TGF-β1 was obtained from Miltenyi Biotec (Bergisch Gladbach, North Rhine-Westphalia, Germany) and SAR405 from Calbiochem (Merck Millipore, Burlington, MA, USA).

Tan M.L., Parkinson E.K., Yap L.F, & Paterson I.C. (2021). Autophagy is deregulated in cancer-associated fibroblasts from oral cancer and is stimulated during the induction of fibroblast senescence by TGF-β1. Scientific Reports, 11, 584.

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Immortalization of Fibroblasts and ADC-TAFs

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Control fibroblasts and ADC-TAFs from randomly selected patients (#5, #13, #37) were immortalised with hTERT as reported [9 (link)]. Unless otherwise indicated, all fibroblast experiments were performed in culture medium containing serum-free high-glucose (4.5 g/l) DMEM supplemented with 1% insulin–transferrin–selenium (ITS) and antibiotics as described [10 (link)]. In some experiments, fibroblasts were stimulated with 2.5 ng/ml recombinant human TGF-β1 (Miltenyi Biotec) at different time points as indicated, which is similar to the average TGF-β1 concentration reported in the bronchoalveolar lavage fluid of lung cancer patients [21 (link)].

Juste-Lanas Y., Díaz-Valdivia N., Llorente A., Ikemori R., Bernardo A., Arshakyan M., Borau C., Ramírez J., Ruffinelli J.C., Nadal E., Reguart N., García-Aznar J.M, & Alcaraz J. (2022). 3D collagen migration patterns reveal a SMAD3-dependent and TGF-β1-independent mechanism of recruitment for tumour-associated fibroblasts in lung adenocarcinoma. British Journal of Cancer, 128(6), 967-981.

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Induction of Regulatory T Cells from Thymocytes

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iTregs were generated as previously described [11] (link), [12] (link). Briefly, CD4 single-positive thymocytes were sorted from 6–8-wk-old TxA23 TCR transgenic mice and stimulated with plate-bound anti-CD3 (1 µg/ml) and anti-CD28 (2 µg/ml) (BD Pharmingen) in 24-well plates (2.5×10⁵ cells/well) in 2 ml complete RPMI media (modified RPMI 1640 supplemented by 10% FBS, 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 10 mM HEPES, 4×10⁻⁷ M 2-ME, 1 mM essential amino acids, and 1 mM sodium pyruvate; all from Sigma Aldrich, MO) supplemented with recombinant human IL-2 (100 U/ml). Recombinant human TGF-β1 (Miltenyi Biotech, Auburn, CA) (5 ng/ml) was added to induce iTregs. Cells were removed from plate-bound antibodies after 48 hours and added to new wells with fresh media supplemented with 100 U/ml recombinant human IL-2. Cells were used after 7 days in culture. The proportion of cells expressing Foxp3 after 7 days of culture was typically 60–80%.

Nguyen T.L., Makhlouf N.T., Anthony B.A., Teague R.M, & DiPaolo R.J. (2014). In Vitro Induced Regulatory T Cells Are Unique from Endogenous Regulatory T Cells and Effective at Suppressing Late Stages of Ongoing Autoimmunity. PLoS ONE, 9(8), e104698.

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Modulation of ELOVL5 and TGF-β Signaling

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Cells were reverse-transfected using Lipofectamine™ RNAiMAX transfection reagent (ThermoFisher Scientific) with non-targeting siRNA (#4390846; Dharmacon), siRNA targeting human ELOVL5 (#s34075; Dharmacon), mouse ELOVL5 (#s87101; Dharmacon) or human smad2 (#s8397; Dharmacon) according to the manufacturer’s instructions. Treatments with 10 µM A922500 (Sigma-Aldrich), 10 µM PF-06424439 (Sigma-Aldrich), 1 µM LY2157299 (Selleck) and 1 µM LY2109761 (Sigma-Aldrich) were performed in complete culture medium. Breast cancer cells were incubated with 5 ng/ml recombinant human TGF-β1 (Miltenyi Biotec) in complete cell culture medium for the indicated times.

Kieu T.L., Pierre L., Derangère V., Perrey S., Truntzer C., Jalil A., Causse S., Groetz E., Dumont A., Guyard L., Arnould L., de Barros J.P., Apetoh L., Rébé C., Limagne E., Jourdan T., Demizieux L., Masson D., Thomas C., Ghiringhelli F, & Rialland M. (2022). Downregulation of Elovl5 promotes breast cancer metastasis through a lipid-droplet accumulation-mediated induction of TGF-β receptors. Cell Death & Disease, 13(9), 758.

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