Transforming growth factor beta tgf β

For MSC differentiation, a 70% subconfluent culture of MSCs from the third passage were used.
The MSCs were placed in basic medium, consisting of Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen Life Technologies), 10% fetal bovine serum (FBS), 1% penicillin, 1% streptomycin and 1% amphotericin B. Specific supplements were added to this basic medium formulation for the differentiation of the MSCs into various mesenchymal lineages[28 (link)]. Adipogenic differentiation was induced with 0.5 μM dexamethasone, 0.5 Mm 3-isobutyl-1-methylxanthine and 0.1 mM indomethacin (Sigma)[29 (link)]. Osteogenic differentiation was achieved with 0.1 μM of dexamethasone, 50 μM ascorbic acid and 10 mM-glycerophosphate (Sigma)[30 (link)]. Chondrogenic differentiation was induced with 50 μM ascorbic acid, 0.1 μM dexamethasone, 10 ng/ml transforming growth factor-beta (TGF-β) (R&D Systems), 40 μg/ml L-proline (Sigma- Aldrich) and 100 μg/ml sodium pyruvate (Wako) [31 (link)]. The differentiation medium was refreshed every 2 days. Differentiation into adipocytes, osteocytes and chondrocytes was confirmed by oil red O, von Kossa and toluidine blue staining, respectively[30 (link)]

CDK8/CDK19 single knockout and double knockout derivatives of human embryonic kidney (HEK) 293 cells (ATCC CRL-1573) were generated as previously described [18 (link)]. All cells were cultured in DMEM (high-glucose) media supplemented with 10% fetal bovine serum (FBS) and Penicillin-Streptomycin-Glutamine (1×) at 37 °C with 5% CO₂ and routinely confirmed to be free of mycoplasma. Microscopic examination was carried out by Olympus CKX41 Inverted Phase Contrast Microscope and the cell images were captured and processed with CellSens standard software.
Human recombinant TNFα (Z00404-50) was obtained from GenScript (Piscataway, NJ, USA). Senexin B and 15w were provided by Senex Biotechnology (Columbia, SC, USA). Didehydro-cortistatin A (dCA) was a gift from Phil S. Baran (Scripps Research Institute, La Jolla, CA, USA). Cmpd3 (CCT251921) and Cmpd4 (MSC2530818) were a gift from Merck KGaA, Darmstadt, Germany. Recombinant human epidermal growth factor (EGF) and transforming growth factor beta (TGFβ) proteins were obtained from R&D Systems, Minneapolis, MN, USA. Other reagents were obtained from MilliporeSigma, St. Louis, MO, USA.

BMDMs were plated in T175 flasks at a cell density of 4 × 10⁶ cells/flask in 35 ml medium and incubated for 5 h. M1 and M2 polarized macrophages were gently washed with warm phosphate buffer saline (PBS) thrice. 35 ml of fresh medium were added and conditioned medium (CM) was collected after 24 h of incubation. CM was passed through a 0.22 µm syringe filter and stored at − 80 °C until used. CM prepared from untreated/non-polarized BMDMs was designated M0 CM.
CM was used at 25, 50 and 100% concentrations as culture medium for MCF7 and MDA-MB468 cells for 48 h. Additionally, cells were treated with recombinant human IFN-γ 10 ng/ml, IL-1β 10 ng/ml, tumor necrosis factor alpha (TNF-α) 10 ng/ml, IL-10 20 ng/ml (all Peprotech) or transforming growth factor beta (TGF-β) 5 ng/ml (R&D, Minneapolis, MN, USA). Cells were incubated for 24 or 48 h before being harvested.