Recombinant human tgf β1

NMuMG mammary epithelial cells were grown in DMEM supplemented with 10% Fetal Bovine serum (FBS) (Wisent), 10 mM HEPES, 1 mM sodium pyruvate, 1 mM L-glutamine, 10 μg/ml insulin and antibiotics as previously described [15 (link)]. HEK293, COS-7, and MDA-MB-231 cells were cultured in DMEM supplemented with 10% FBS and antibiotics in a humidified incubator at 37°C with 5% CO_2. HEK293 and COS-7 cells were transfected with the indicated constructs using linear polyethylenimine (PEI) (Polysciences) at a 1:8 ratio (cDNA:PEI) following the manufacturer’s instructions. For siRNA transfections, MDA-MB-231 cells were transfected with siRNAs targeting RSK1/2 or a scrambled control siRNA duplex using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s protocol. The final concentration of both siRNAs was 30 nM. Thirty hours post-transfection, cells were serum-starved for 18h and stimulated for 30 min with either 20% FBS, 200 nM PMA (phorbol-12-myristate-13-acetate, Cell Signaling) or 5 ng/ml recombinant Human TGF-β1 (Invitrogen). For PMA and BI-D1870 (Stemgent) treatments, cells were treated for 30min with PMA (200nM) and BI-D1870 (20nM) following a pre-treatment with BI-D1870 (20nM) for 1h.

The U87MG cell line was purchased from the American Type Culture Collection (ATCC, USA). The SNB19, U251, LN229, LN308 and U343 cell lines were obtained from the Chinese Academia Sinica Cell Repository. Isolated from a Chinese patient, the TJ905 cell line was maintained in our lab. The human non-tumoral control cell line UC2 was established by immortalizing astrocytes. All these cells were authenticated and tested for mycoplasma contamination. They were maintained in Dulbecco’s modified Eagle’s medium containing 10% foetal bovine serum (Gibco, USA) at 37 °C in a humidified incubator with 5% CO₂. Recombinant human TGF-β1 was purchased from Invitrogen (USA), and LY2109761 (TGFβR2 inhibitor) was purchased from Selleck (China).

Flow-cytometry sorted Ly-6C^- and Ly-6C⁺ CD4 T_N cells from LNs of C57BL/6 Foxp3-GFP mice were stimulated as described above with immobilized anti-CD3 and anti-CD28 Abs in the presence or absence of exogenous recombinant human TGFβ1 (Invitrogen, 4 µg/mL). Supernatants were recovered 24 hr later and cytokines were quantified by MSD multi-array U-PLEX assays (IFN-γ, IL-4, IL-17A/F and IL-10; Meso Scale Discovery, Rockville, MD) according to the manufacturer’s instructions.

After excision, the human endometriotic tissue was rinsed with phosphate buffered saline (PBS). The endometrial lining was detached from the myometrium, minced in Hank’s balanced salt solution, and then digested with collagenase II (Solarbio, Beijing, China) at 37 °C for 2 h. Follwing this, they were centrifuged, rinsed, and resuspended in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium (Solarbio) containing 10% fetal bovine serum (FBS; Solarbio), 100 U/mL penicillin (Solarbio), and 100 mg/mL streptomycin (Solarbio). The cell suspension was then filtered through a 100 mm cell strainer (BD Falcon, Franklin Lakes, NJ, USA). Following this, cells were centrifuged, resuspended, and cultured in DMEM medium in a 5% CO₂ incubator at 37 °C. At confluence, endometriotic cyst stromal cells were treated with 10 ng/mL recombinant human TGF-β1 (catalog number: PHG9214; Invitrogen, Carisbad, CA, USA) for 48 h.

To change the contractility of the cytoskeleton, the treatment of the samples with three different drugs was performed starting at day 4 after seeding until the end of the experiment: myosin II inhibitor Blebbistatin to inhibit the myosin–actin contractility; TGF-β1 to enhance the contractility and stabilize the cytoskeleton or Latrunculin A to inhibit actin assembly. (−)-Blebbistatin (Sigma-Aldrich Chemie GmbH) was added to the growth media at a final concentration of 2 µM with 0.1% DMSO (Sigma-Aldrich Chemie GmbH). The recombinant human TGF-β1 (Invitrogen, MD, USA) was added at a final concentration of 1 ng/mL. The actin monomer-binding toxin Latrunculin A (Millipore, Darmstadt, Germany) was added at a final concentration of 20 nM with 0.1% DMSO. To exclude the effect of the solvent DMSO, additional samples were treated with 0.1% DMSO in the same period.