Tgf β

Using A549 cells, we treated TGF-β (Sigma-Aldrich, #T7039). Before the treatment of TGF-β, cells were starved with 0.1% FBS for 24 h^{43 (link)}. After starvation, we treated 5 ng/ml TGF-β diluted with 2 mg/ml Bovine Serum Albumin (BSA, Sigma-Aldrich, #A9647) for 48 h with 0.1% FBS. In the case of the combination with TGF-β and Kv3.4 overexpression, Kv3.4-overexpressed cells were also starved with 0.1% FBS with G-418 for 24 h. And then, 2 ng/ml of TGF-β was treated in the cells for 24 h^{44 (link)}. After the treatment, the cells were prepared for RNA or protein extraction.

A549 and HEK293T cell lines were maintained in high glucose DMEM (Sigma-Aldrich) supplemented with 2 mM L-glutamine, 10% FCS and antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin), and 1:100 Biomyc3 anti-mycoplasma antibiotic solution (Biological Industries, Beit HaEmek, Israel). Cells were grown at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. For TGFβ stimulation, cells were cultured with 5 ng/ml recombinant human TGFβ (PeproRTech) in cell medium. If cells were stimulated for more than 24 h with TGFβ, the TGFβ in the medium was refreshed every 24 h. For EMT induction in HMLE-Twist-ER cells, cells were treated with 10 nM 4-hydroxy tamoxifen (OHT) (Sigma-Aldrich, H7904) for the indicated number of days. During treatment, the medium was refreshed every 2 days.

Human hepatic stellate cell line, LX-2, was used as cell model. LX-2 cells seeded in 6-well plate were cultured at 37°C in room air in incubator in Dulbecco's modified Eagle medium (Gibco, USA) with 5% CO₂ supplemented with 10% fetal bovine serum (Gibco, USA), 100 U/ml penicillin and 100 mg/ml streptomycin. LX-2 cells were randomly divided into groups: Control, LX-2 cells were cultured under normal condition; TGF-β+PBS, LX-2 cells were stimulated by 2 ng/ml TGF-β1 (Sigma, USA) for 72 hours; TGF-β+3MA, TGF-β+Rapa, TGF-β+SN50, TGF-β+PMA+3MA, after treating with TGF-β1 48 hours, LX-2 cells were cultured with 3MA (1 mg/ml, Sigma), rapamycin (200 μg/ml; Sigma, USA), SN50 (80 μg/ml), Phorbol 12-myristate 13-acetate (PMA, 30 ng/ml, Sigma, USA) and 3MA (1 mg/ml, Sigma, USA), respectively. Cells were collected after culturing 72 hours.

Breast cancer cell lines MDA‐MB‐231 were purchased from the Chinese Academy of Sciences, and cultured in L15 (Zhong Qiao Xin Zhou) medium supplemented with 10% Fetal Bovine Serum (FBS) and penicillin (100 IU/ml) /streptomycin (100 μg/ml) (Gibco) in a humidified incubator at 37°C. For testing the effect of PFD on TGF‐β‐induced activation of Smad signalling pathway and expression of EMT‐related genes, the breast cancer cells were pre‐treated with 5 ng/ml TGF‐β (Millipore Sigma) for 24 h, followed by addition of PFD (4 mM) or LY2109761 (2 μM) for another 24 h.

The previously-described adherent cells were detached and labeled with CD19, CD34, CD73, CD90, CD45 and CD105 fluorescent conjugated antibodies (Becton, Dickinson and Company, NJ, USA). After evaluation by assessing the percentages of CD19(−), CD34(−), CD73(+), CD90(+), CD45(−) and CD105(+) cells, results of evaluation are displayed in Supplementary Figure S1A. The adipogenic, osteogenic and chrondrogenic differentiation abilities of the cells were also assessed in Figure S1C. For the adipogenic assay, 10000 MSCs were seeded into 24-well plates (per well) and cultured in DMEM (Sigma) containing 10% FBS and 100 mm/L indometacin, 10 μg/mL insulin, 0.5 mmol/L IBMX and 100 nmol/L dexamethasone (all Sigma) for every 3 day, then the cells were then stained with Oil Red O. For the osteogenic assay, 5000 MSCs (per well) were seeded into 24-well plates and cultured in DMEM (Sigma, City, CA, USA) containing 10% FBS and 0.1 μmol/L dexamethasone, 50 μmol/L ascorbic acid and 10 mmol/L β-glycerophosphate (all Sigma). After 4 weeks, the cells were stained with alizarin red (Sigma).For the chrondrogenic assay, 10000 MSCs were seeded into 24-well plates (per well) and cultured in DMEM (Sigma) containing 10% FBS and 10 ng/mL TGF-β, 10 μg/mL insulin and 100 nmol/L dexamethasone (all Sigma) for every 3 day, then the cells were then stained with toluidine blue.