Uo126

Dulbecco's modified Eagle's medium (DMEM), RPMI1640 and the antibiotics were purchased from Life Technologies (Rockville, MD, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA). Twenty four-well invasion chambers (8 μm pore) were obtained from Becton-Dickinson (San Diego, CA). ZER was a generous gift from Dr. Murakami Akira (Kyoto University, Japan). LY2109761 was purchased from Selleck Chemicals (Houston, TX, USA). UO126 (a MEK1/2 inhibitor) were purchased from Tocris (Ellisville, MO, USA). SIS3 (a smad3 inhibitor) was purchased from Calbiochem (San Diego, CA, USA). The secondary horseradish peroxidase (HRP)-conjugated and mouse monoclonal anti-β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Mouse monoclonal anti-human Ki-67 antigen was purchased from Dako (Cambridgeshire, UK). Rabbit monoclonal anti-FN and smad3 (total and phospho-form) antibodies were purchased from Epitomics (Burlingame, CA, USA). Rabbit polyclonal ERK1/2 (total and phospho-form) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Recombinant Human TGF-β1 was purchased from R&D Systems (Minneapolis, MN, USA). The ECL prime reagents were from Amersham (Buckinghamshire, UK).

NR8383 cells were first treated with one of the following: CX3CL1 protein (R&D Systems, Minneapolis, MN, USA), anti-CX3CR1 antibody (R&D Systems, Minneapolis, MN, USA), MFG-E8 protein (R&D Systems, Minneapolis, MN, USA), Bay11–7082 (InvivoGen, San Diego, CA, USA) or UO126 (Tocris Bioscience, Bristol, UK). Thereafter, NR8383 cells were washed with 5% PBS solution twice. Subsequently, the NR8383 cells were incubated with PKH−26 (Sigma-Aldrich, St. Louis, MO, USA) labeled Ida-ATRA-NB4 cells (2 × 10⁶) for 30 min before determining the amount of phagocytosis using a BD FACScan [24 (link)]. A subset of the Ida-ATRA-NB4 cells was treated with anti-CX3CL1 (R&D Systems, Minneapolis, MN, USA) before labeling with PKH−26. The results were expressed either as the percentage (%) of NR8383 cells with phagocytic activity in engulfing apoptotic cells or as a phagocytosis index that indicates a fold increase relative to the phagocytic activity of untreated NR8383 cells.