Tgf β

ELISA kits were used to assess the expression of selected proteins in mouse plasma and tumor homogenates. Assays were performed according to the manufacturer’s protocols, and absorbance values were measured using a plate reader (BioTek Instruments, Winooski, VT, USA). The expression of the following proteins was measured in plasma: C-C motif chemokine ligand 2 (CCL2), TGF-β (eBioscience, Amsterdam, The Netherlands), IL-6 (Wuhan EIAab Science Co, Ltd., Wuhan, China), OPN (Thermo Fisher Scientific, Waltham, MA, USA), and vascular endothelial growth factor (VEGF; Invitrogen, Carlsbad, CA, USA). The expression of the following proteins was measured in tumor samples: CCL2 (eBioscience, Amsterdam, The Netherlands), fibroblast growth factor 23 (FGF-23; Abcam, Cambridge, UK), IL-6 (Wuhan EIAab Science Co, Ltd., Wuhan, China), OPN (Thermo Fisher Scientific, Waltham, MA, USA), SDF-1, TGF-β, and VEGF (Invitrogen, Carlsbad, CA, USA). The results obtained were analyzed using the CurveExpert ver. 1.4 software.

To determine the biological activity of TGF-β, NMuMG cells were used. Briefly, 5 × 10³ of NMuMG cells per well were treated with PBS, 2 ng/ml TGF-β (Gibco, United States), and cryogel released TGF-β (day 3). Cells were incubated at 37°C for 30–48 h and MTT assay was performed. 10 μl MTT solution was added to each well and incubated for 4–6 h. Finally, 100 μl of isopropanol in 0.04 M HCl was added to each well and the absorbance was measured at 570 nm using a spectrophotometer (Varioskan Flash, Thermo Scientific, United States).

In order to assess ability of dolphin lymphocytes to respond to a Th1, Th2, or Treg stimulus, dolphin PBMCs (2 × 10⁶ cells/ml) were incubated in 96 well flat bottom plates with human recombinant cytokines at concentrations of 0 (unstimulated), 1, 10, and 25 pg/ml for 24 h. To assess a Th1 response, cells were stimulated with IL-12 (Millipore Sigma, Burlington, MA 01803, USA) and IFNγ (Thermo Fisher Scientific, Grand Island, NY 14072, USA) and analyzed for IFNγ expression. To stimulate a Th2 response, cells were stimulated with IL-4 (Millipore Sigma, Burlington, MA 01803, USA) and analyzed for IL-4 and IL-13 expression. To assess a Treg response, cells were stimulated with IL-2 (Thermo Fisher Scientific, Grand Island, NY 14072, USA) and TGFβ (Thermo Fisher Scientific, Grand Island, NY 14072, USA) and analyzed for TGFβ and IL-10 expression. After a 24 h incubation at 37°C and 5% CO₂, cells were collected from the plates, centrifuged at 220 g for 10 min, re-suspended in RNAlater solution (Thermo Fisher Scientific, Grand Island, NY 14072, USA) and stored at 4°C for up to 1 month. RNAlater samples were then moved to −20°C for long-term storage.

EO771 mammary tumor cells (CH3 BioSystems, Amherst, NY, USA) [47 (link)] and astrocytes (Cell Biologics, Chicago, IL, USA) were grown in DMEM (Corning, Inc., Corning, NY, USA), MLO-A5 osteocyte-like cells (obtained from Dr. L. Bonewald at Indiana University, Indianapolis, IN, USA), and bone marrow-derived MSCs (harvested from C57BL/6 mice) were cultured in αMEM (Gibco, Carlsbad, CA, USA). The culture media were supplemented with 10% fetal bovine serum and antibiotics (50 units/mL penicillin, and 50 µg/mL streptomycin; Life Technologies, Carlsbad, CA, USA), and cells were incubated at 37 °C with 5% CO₂. To test the effect of TGFβ, EO771 cells were treated with TGFβ (500 ng/mL, Thermo Fisher Scientific, Waltham, MA, USA) for 1 day.
CM was prepared from MLO-A5 osteocytes and astrocytes at ~80% confluence after 24 h incubation and an Amicon filter unit with a cutoff mass at 3 kDa (Sigma-Aldrich, St. Louis, MO, USA) was used to remove microparticles and condense it by 10-fold. Cellular proliferation was examined using an MTT assay, and a wound-healing scratch assay and a Transwell invasion assay were conducted to evaluate cell motility and invasion capability, respectively, using the procedure previously described [48 (link)].