Anti tgf β1 neutralizing antibody

Transwell co-culture experiments were performed in 24-well plates with 0.4 μm pore sizes in inner wells (Greiner, Frickenhausen, Germany) to physically separate CD8⁺ T cells and SKOV3/A2780 cells. Approximately 2 × 10⁵ SKOV3/A2780 cells per well were grown in the outer wells using 1.5 mL 1640-medium containing 10% human AB serum. Next, 6 × 10⁵ isolated human CD8⁺ T cells were added into the inner wells in 500 μL of the same medium. For TGF-β1 neutralization experiments, 10 μg/mL anti-TGF-β1 neutralizing antibody (R&D Systems, Minneapolis, MN, USA) was added to the SKOV3/A2780 co-culture system to block TGF-β1 function. After 5 days incubation, CD8⁺ T cells were harvested and washed with PBS. Approximately 1 × 10⁶ cells were collected for flow cytometry analysis, and 2 × 10⁶ cells were collected for western blot analysis. The remaining cells were dispensed into 96-well plates at 4×10⁴ cells per well and stimulated with 1 mg/mL soluble anti-CD3 and anti-CD28 antibodies (eBioscience, San Diego, CA, USA). After 6 and 24 h stimulation, CD8⁺ T cells were collected for evaluation of cytokine mRNA expression levels by RT-PCR.

5(S), 6(R)-Lipoxin A4 (LXA4) and 5(S), 6(R)-7-trihydroxymethyl Heptanoate (BML-111) were purchased from Cayman Chemical (Ann Arbor, MI, USA). Antibodies against E-cadherin, N-cadherin, Vimentin, and Snail were obtained from Cell Signaling Technology (Boston, MA, USA). The antibodies against FPRL1 and 15-LOX were purchased from Abcam (Cambridge, UK). The anti-TGF-β1 neutralizing antibody was obtained from R&D Systems (Minneapolis, MN, USA), and recombinant human TGF-β1 (rhTGF-β1) was obtained from PeproTech (Rocky Hill, NJ, USA). Anti-β-actin was purchased from Sigma-Aldrich (St. Louis, MO, USA).

SC (1 × 10⁵/mL, 12-well plates) were pre-treated for 2 h with 10 µM COX-2 inhibitor (CAS 181696-33-3, Calbiochem, San Diego, CA, USA), 10 µM TGF-βR1/ALK4/5/7 inhibitor (SB431542, MilliporeSigma), 10 µM MAPK/ERK inhibitor (UO126, Cell Signaling Technology) or medium served as a control. Medium was removed and SC were treated with tumor-conditioned medium (10% v/v), control medium or rTGF-β1 (100 pg/mL, Cell Signaling Technology, Danvers, MA, USA) for 24 h. To block the TGF-β1 activity, 10 μg/mL anti-TGF-β1-neutralizing antibody (R&D System, Minneapolis, MN, USA) was added at the beginning of stimulation. Medium was removed and replaced with complete medium (2% FBS) for additional 24-h incubation. Cell-free supernatants were collected by centrifugation at 700× g for 5 min. PGE2 concentration (Cayman Chemical, USA) was measured in supernatants by ELISA.