Htgf β1

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom, United States

HTGF-β1 is a recombinant human transforming growth factor beta 1 (TGF-β1) protein. TGF-β1 is a multifunctional cytokine that regulates cell growth, differentiation, and other cellular functions.

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13 protocols using htgf β1

Chondrogenic Differentiation of MSCs

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MSCs were kindly provided by the research group of Prof. Galderisi of the Department Experimental Medicine of University of Campania “Luigi Vanvitelli.” Chondrogenic differentiation was performed in GM10 based scaffolds. Briefly, the dried cross‐linked biomaterials were placed in a standard 24‐well culture plate, 5.0 × 10⁴ MSCs aliquots were suspended in 10 μl of chondrogenic culture medium composed by: DMEM (Dulbecco's‐modified Eagle's medium, Gibco, Invitrogen) with 10% vol/vol of FBS (Fetal Bovine Serum, Gibco, MA, Invitrogen), 50 nM ascorbate‐2‐phosphate (Sigma‐Aldrich, MO, USA), 0.1 mM dexamethasone (Sigma‐Aldrich, Saint Louis, MO, USA), and 10 ng/ml of human transforming growth factor (hTGF)‐β1 (Preprotech, UK) and seeded in GM10, or GM10 + BC, or GM10 + CS based biomaterials. Once the culture medium containing the cells was completely absorbed by the hydrogels, other 500 μl of medium were added to each well to cover the scaffold (matrix). The MSCs containing biomaterials were maintained at 37°C in a humidified atmosphere with 5% vol/vol CO₂ until 21 days, replacing the culture medium every 48 h.

Vassallo V., Tsianaka A., Alessio N., Grübel J., Cammarota M., Tovar G.E., Southan A, & Schiraldi C. (2022). Evaluation of novel biomaterials for cartilage regeneration based on gelatin methacryloyl interpenetrated with extractive chondroitin sulfate or unsulfated biotechnological chondroitin. Journal of Biomedical Materials Research. Part a, 110(6), 1210-1223.

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Wnt/β-catenin Signaling Regulation in LR-MSCs

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Human LR-MSCs were treated with or without h TGF-β1 (Cell Signaling Technology, Beverly, MA) at 10 ng/ml for indicated periods of time, and then harvested for analysis of the myofibroblast markers and the key components of Wnt/β-catenin signaling. A small-molecule inhibitor, ICG-001, which specifically disrupts β-catenin signaling in a CBP-dependent fashion has been reported in previous literature^{26 (link)}. Both human and mouse LR-MSCs were pretreated with ICG-001 (Selleckchem, Houston, TX) for 1 h, followed by incubation with 10 ng/ml h TGF-β1 or TGF-β1 (PeproTech, Rocky Hill, NJ) for 24 h. The cells were collected for Q-PCR, Western blot analysis, and immunofluorescence staining.

Cao H., Wang C., Chen X., Hou J., Xiang Z., Shen Y, & Han X. (2018). Inhibition of Wnt/β-catenin signaling suppresses myofibroblast differentiation of lung resident mesenchymal stem cells and pulmonary fibrosis. Scientific Reports, 8, 13644.

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3D Chondrogenic Differentiation Protocol

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Chondrogenic differentiation was performed in 3D culture. Briefly, 1x10⁵ cells were seeded as a pellet in 96 round bottom multi-wells and cultured in achondrogenic medium composed of DMEM, 1% FBS, 50 nM ascorbate-2-phosphate (Sigma-Aldrich, MO, USA), 0.1 mM dexamethasone (Sigma-Aldrich, Saint Louis, MO, USA), and 10 ng/mL human transforming growth factor (hTGF)-β1 (Preprotech, UK). The medium was replaced every 3 days. Differentiation was performed either in presence or in absence of the above indicated hyaluronan complexes.
After 21 days, Safranin O staining was performed to detect glycosaminoglycan formation on the cell surfaces, (Sigma-Aldrich, MO, USA) In short, cell pellets were fixed in cold (4°C) acetone:methanol solution for 60 min and then included in cryostat embedding medium (Bioptica, Italy) for cryosectioning. Pellet sections were incubated at room temperature in 1% Safranin O solution for 30 min followed by three rinses in 3% acetic acid for 2 min each. After rinsing in deionized water for 2 min, the surfaces were allowed to dry for imaging.

Alessio N., Stellavato A., Squillaro T., Del Gaudio S., Di Bernardo G., Peluso G., De Rosa M., Schiraldi C, & Galderisi U. (2018). Hybrid complexes of high and low molecular weight hyaluronan delay in vitro replicative senescence of mesenchymal stromal cells: a pilot study for future therapeutic application. Aging (Albany NY), 10(7), 1575-1585.

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Chondrogenic Differentiation of MSCs

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MSCs were seeded as a pellet in 96 round-bottom multi-wells and cultured in a chondrogenic medium composed of DMEM, 1% FBS, 50 nM ascorbate-2-phosphate (Sigma-Aldrich, St. Louis, MO, USA), 0.1 mM dexamethasone (Sigma-Aldrich, MO, USA), and 10 ng/mL human transforming growth factor (hTGF)-β1 (PeproTech, London, UK). After 21 days, Safranin O staining was performed to detect glycosaminoglycan formation on the cell surfaces.

Alessio N., Acar M.B., Demirsoy I.H., Squillaro T., Siniscalco D., Bernardo G.D., Peluso G., Özcan S, & Galderisi U. (2020). Obesity is associated with senescence of mesenchymal stromal cells derived from bone marrow, subcutaneous and visceral fat of young mice. Aging (Albany NY), 12(13), 12609-12621.

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Differentiation of Murine T Helper 9 Cells

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Naïve CD4 T cells were isolated from mouse spleens using CD4⁺CD62L⁺ T cell isolation kit provided by the supplier (Miltenyi Biotec). Cells were cultured in complete RPMI 1640 media on anti-CD3 (2 Units/ml 145–2C11; BioXCell) coated-plates and soluble anti-CD28 (2.5 μg/ml; BD Pharmingen) under T_H9 polarizing conditions including: hTGF- β1 (2 ng/ml), IL-4 (20 ng/ml), hIL-2 (50 U/ml), anti-IL-10R (10 μg/ml) and anti-IFN-γ (10 μg/ml) (All cytokines were obtained from PeproTech and antibodies were obtained from BioXcell). On day 3, cells were expanded into fresh media containing the original concentrations of cytokines in the absence of co-stimulatory signals for an additional 2 days. On day 5, mature T_H9 cells were harvested for further analysis.

Kharwadkar R., Ulrich B.J., Qayum A.A., Koh B., Licona-Limon P., Flavell R.A, & Kaplan M. (2020). Expression efficiency of multiple Il9 reporter alleles is determined by cell lineage. ImmunoHorizons, 4(5), 282-291.

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TGF-β1 and BI6727 Induced Fibroblast Differentiation

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NIH/3T3 or primary cells were starved in DMEM without FBS for 24 h, and then cultured in DMEM with 5% FBS and 5 nM hTGF-β1 (Peprotech, 500-M66) or 10 nM BI6727 (Selleck, S2235) for three days. Immunofluorescence was performed as previously described [44 (link)].

Yu J., Xu H., Cui J., Chen S., Zhang H., Zou Y., Zhao J., Le S., Jiang L., Chen Z., Liu H., Zhang D., Xia J, & Wu J. (2020). PLK1 Inhibition alleviates transplant-associated obliterative bronchiolitis by suppressing myofibroblast differentiation. Aging (Albany NY), 12(12), 11636-11652.

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Isolation and Differentiation of CD4+ T Cells

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Total and CD4⁺ T cells were isolated from the spleens and LNs using the Mouse pan T Cell Enrichment Kit or the Mouse CD4⁺ T Cell Enrichment Kit, respectively (both STEMCELL Technologies; cat. nos 19852 and 19751). CD4⁺ T cells were stimulated with 1 μg ml⁻¹ plate-bound anti-CD3 (clone 2C11) plus 1 μg ml⁻¹ anti-CD28 antibodies (clone 37.51; both Bio X Cell). For differentiation of naive CD4⁺ T cells into T_H1, T_H17 and T_H2 cells, T cells were polarized for 3 days with 10 ng ml⁻¹ IL-12 (PeproTech) and 2 μg ml⁻¹ anti-IL-4 (eBioscience) for T_H1; 20 ng ml⁻¹ IL-6 (PeproTech), 0.5 ng ml⁻¹ hTGFβ1 (PeproTech), 2 μg ml⁻¹ anti-IL-4 (clone 11B11) and 2 μg ml⁻¹ anti-IFNγ (clone XMG1.2; both eBioscience) for T_H17 and 100 ng ml⁻¹ IL-4 (PeproTech), 5 μg ml⁻¹ anti-IL-12 and 20 μg ml⁻¹ anti-IFNγ (both eBioscience) for T_H2 cells in IMDM or RPMI medium (both Cellgro). Both media contained 2 mM L-glutamine, 50 mM 2-ME, 100 U ml⁻¹ penicillin/streptomycin and 10% FCS. For cytokine expression, cells were (re-)stimulated with 1 μM ionomycin plus 20 nM phorbol myristate acetate (both Calbiochem) for 6 h in the presence of brefeldin A (BioLegend) and analysed by flow cytometry as described below.

Vaeth M., Yang J., Yamashita M., Zee I., Eckstein M., Knosp C., Kaufmann U., Karoly Jani P., Lacruz R.S., Flockerzi V., Kacskovics I., Prakriya M, & Feske S. (2017). ORAI2 modulates store-operated calcium entry and T cell-mediated immunity. Nature Communications, 8, 14714.

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In Vivo T Cell Activation Assay

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All animal studies were performed according to National Institutes of Health (NIH) guidelines for the use and care of live animals and were approved by the Animal Care and Use Committees of the National Cancer Institute (NCI) under the protocol 20-073. C57BL/6 CD4-Cre transgenic, RAG^−/− mice and Ifngr KO (JAX:003288) mice were obtained from the Jackson Laboratory and bred in our institute animal facility under specific pathogen–free conditions. Sptlc1^foxl/flox mice were generated in our laboratory as previously described (34 (link)). Sptlc1^foxl/floxCD4-Cre (KO) conditional KO mice were generated by crossing Sptlc1^floxl/flox mice to CD4-Cre transgenic mice. Sptlc1^foxl/flox or Sptlc1^+/+CD4-Cre were used as control mice (WT). All mice used for experiments were aged 8 to 12 weeks, and both female and male mice were used for experiments. Primers for qPCR were obtained from IDT (Integrated DNA Technologies, USA). Cytokines mIL-4, mIL-12, hTGF-β1, IL-7, and IL-1β were purchased from PeproTech, USA; hIL-2 was obtained from R&D Systems; and IL-6 and IL-23 were purchased from BioLegend, USA. Antibodies for activation and cytokine neutralization were procured from Bio X Cell, USA. All chemicals were purchased from Sigma-Aldrich unless otherwise mentioned.

Abimannan T., Parthibane V., Le S.H., Vijaykrishna N., Fox S.D., Karim B., Kunduri G., Blankenberg D., Andresson T., Bamba T., Acharya U, & Acharya J.K. (2024). Sphingolipid biosynthesis is essential for metabolic rewiring during TH17 cell differentiation. Science Advances, 10(17), eadk1045.

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Differentiation of Pathogenic and Non-pathogenic Th17 Cells

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CD4⁺ T cells were isolated from the spleen and submandibular, axillar, inguinal, and mesenterial lymph nodes of mice by negative enrichment using a MagniSort mouse CD4⁺ T‐cell enrichment kit (eBioscience) and stimulated with 0.25 μg/ml plate‐bound anti‐CD3 (clone 2C11) and 1 μg/ml anti‐CD28 (clone 37.51; both Bio X Cell) antibodies in the presence of 2 μg/ml anti‐IL‐4 (clone 11B11) and 2 μg/ml anti‐IFN‐γ (clone XMG1.2; both eBioscience). For the differentiation into non‐pathogenic Th17 cells, CD4⁺ T cells were cultured in the presence of 20 ng/ml IL‐6 (Peprotech) and 0.5 ng/ ml hTGFβ1 (PeproTech) for 2 or 3 days. For the differentiation into pathogenic Th17 cells, CD4⁺ T cells were cultured in the presence of 20 ng/ml IL‐6 (Peprotech), 20 ng/ml IL‐1β (Peprotech), and 20 ng/ml IL‐23 (eBioscience) for 2 or 3 days.

Kahlfuss S., Kaufmann U., Concepcion A.R., Noyer L., Raphael D., Vaeth M., Yang J., Pancholi P., Maus M., Muller J., Kozhaya L., Khodadadi‐Jamayran A., Sun Z., Shaw P., Unutmaz D., Stathopulos P.B., Feist C., Cameron S.B., Turvey S.E, & Feske S. (2020). STIM1‐mediated calcium influx controls antifungal immunity and the metabolic function of non‐pathogenic Th17 cells. EMBO Molecular Medicine, 12(8), e11592.

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Osteoblast Signaling Pathway Analysis

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Primary osteoblasts stimulated with either hBMP2 (100 ng/ml) or hTGFβ1 (1 ng/ml; PeproTech) following starvation in serum-free medium for 6 h were lysed in lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing proteinase inhibitor (Roche). Vertebra samples (L5) were pulverized using a mortar and pestle, and suspended in the lysis buffer. Total protein amount was determined by BCA assay (Pierce, Waltham, MA, USA) and immunoblotting was performed with antibodies against pSmad1/5/8 (1:1000; #9511s, Cell Signaling Technology), Smad1/5/8 (1:1000; sc-6031, Santa Cruz Biotechnology, Dallas, TX, USA), pSmad3 (1:1000; #9520, Cell Signaling Technology), Smad3 (1:1000; #9523, Cell Signaling Technology), menin (1:10 000; A300-105A, Bethyl Laboratories, Montgomery, TX, USA), and β-actin (1:1000; sc-1616, Santa Cruz Biotechnology). Band intensity was analyzed using ImageJ software (NIH, Bethesda, MD, USA).

Liu P., Lee S., Knoll J., Rauch A., Ostermay S., Luther J., Malkusch N., Lerner U.H., Zaiss M.M., Neven M., Wittig R., Rauner M., David J.P., Bertolino P., Zhang C.X, & Tuckermann J.P. (2017). Loss of menin in osteoblast lineage affects osteocyte–osteoclast crosstalk causing osteoporosis. Cell Death and Differentiation, 24(4), 672-682.

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