Tgf beta 1

Antibodies information can be found in Supplementary Table S1. Lipofectamine® 3000, RNAiMAX, MessengerMAX, Opti-MEM® reduced serum media, and SYBR® Select Master Mix, were obtained from Invitrogen® (Thermo Fisher, Life-Technologies, Carlsbad, CA, USA). RPMI-1640, DMEM medium, and FBS were obtained from Gibco® (Thermo Fisher, Life-Technologies, Carlsbad, CA, USA). PrimeScript® RT reagent Kit with gDNA Eraser was obtained from Takara bio (Dalian, China). TGF-Beta1 was purchased from PeproTech® (London, UK). mMESSAGE mMACHINE® T7 ULTRA Transcription Kit was obtained from Ambion® (Thermo Fisher, Life-Technologies, Carlsbad, CA, USA) LY2109761(Cat.#S2704) was obtained from Selleck (Houston, TX, USA)
SimpleChIP® Plus Enzymatic Chromatin IP Kit-Magnetic Beads (#9005) were purchased from Cell Signaling Technology (Danvers, MA, USA), and Pierce® Crosslink Magnetic IP/Co-IP Kit(#88805) came from Thermo Fisher Scientific (Waltham, MA, USA). siRNAs for RbBP5, SNAI1, CBP, SMAD2 were synthesized by Ribo Bio(Guangzhou, China). For their sequences, see the Supplementary Table S2.

Hepatocyte cell lines used in this work (MMH/E14 and WT/3A) are standardly grown in RPMI (Gibco) medium supplemented with 10% FBS, 10μg/ml Insulin, 50ng/ml EGF and 30ng/ml IGF-2, on collagen I (Transduction Laboratories, Lexington, UK) coated dishes (Falcon-BD, Franklin Lakes, NJ, USA). MeT-5A lung mesothelial cell line (ATCC® CRL-9444™) is grown in DMEM (Gibco) supplemented with 10% FBS on plastic (Falcon-Bd Franklin Lakes, NJ, USA).
For treatments the media have been supplemented, where indicated, with 10ng/ml of TGFbeta1 (PeproTech Inc, Rocky Hill, NJ), 200ng/ml of Recombinant Murine Wnt3a (315–20, PeproTech Inc., Rocky Hill, NJ, USA), 100ng/ml FGF-1 of Recombinant Human Fibroblast Growth Factor Acidic (13241–013, Gibco, ThermoFisher Scientific Inc., MA, USA), 100ng/ml of FGF-2 Recombinant Mouse Fibroblast Growth Factor Basic (12343623, ImmunoTools GmbH, Germany), 2μM of Src inhibitor PP2 (Calbiochem, Merck Chemicals Ltd. Beeston, Nottingham, UK), 0.5μM of Src inhibitor SU6656 (Cayman Chemical, Ann Arbor, MI, USA), 10μM of PI3K inhibitor LY294002 (Calbiochem, Merck Chemicals Ltd. Beeston, Nottingham, UK) and 5μM of TGFβ receptor I/II inhibitor LY2109761 (Selleckchem,USA).

MSCs were pre-seeded in each scaffold with 1 × 10⁷ cells/mL in a 24-well culture plate, followed by incubation for four hours in an incubator at 37 °C, 5% CO₂. After that, all of the scaffolds were induced to undergo chondrogenic differentiation. Specifically, the constructs were cultured in basic medium as the control consisted of DMEM-high glucose supplemented with 100 nM dexamethasone, 50 μg/mL ascorbic acid, 100 μg/mL sodium pyruvate, 40 μg/mL proline, and 50 mg/mL ITS + 1 liquid media supplement. The chondrogenic differentiation medium was the basic medium with 10 ng/mL recombinant human transforming growth factor-beta 1 (TGF-beta 1; PeproTech Inc., Rocky Hill, NJ, USA). At days 7, 14, 21, and 28, the samples were harvested for subsequent characterization.