Macrophage-Associated Mesenchymal Stem Cells Figure 7. Macrophages induce activation of the c-Jun NH2-terminal kinase pathway in MSCs. To validate the macrophage-induced activation of the c-Jun NH2-terminal kinase (JNK) signaling pathway in MSCs, protein levels of phosphorylated downstream components were assessed by immunoblotting. Exposure to CM from dU937 macrophages induced activation of JNK1, 2, 3 after 15 minutes (A). An increase in the expression of phospho-c-Jun and decrease in the expression of ATF-2 was observed after a 30-minute treatment with dU937 CM (B). These results suggest that in addition to its control of cellular differentiation, proliferation, and apoptosis, the AP-1 transcription factors may also play a role in cellular chemotaxis (C). doi:10.1371/journal.pone.0035036.g007 streptomycin, and 20 nM phorbol-myristate acetate (PMA) (Sigma-Aldrich, St. Louis, MO). Cells were differentiated for 4 days and then used for experiments. Immunoblotting Cells were trypsinized to remove adherent cells from the tissue culture flask. The cell pellets were washed with PBS and re- suspended in 150 mL 1x radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors. Each sample was sonicated for 10– 15 seconds to shear the DNA and reduce sample viscosity. The samples were cooled on ice for 30 minutes, and centrifuged at 14,000 RPM for 20 minutes at 4uC. A Bradford Assay determined protein concentration and lysates were separated on an 8% SDS- polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane for 1 hour. The nitrocellulose membrane was washed with 1x Tris Buffered Saline (TBS) for 5 minutes followed by incubation in blocking buffer for 1 hour at room temperature. The membrane was washed 3 times for 5 minutes each in TBS/T (1x TBS + 0.1% Tween-20) was buffer. The membrane was incubated in primary antibody (at the appropriate dilution) overnight at 4uC with gentle agitation. The membrane was washed 3 times for 5 minutes each in TBS/T. The membrane was incubated with HRP-conjugated secondary antibody (1:2,000) and HRP-conju- gated anti-biotin antibody (1:1,000) (Cell Signaling Technology, Danvers, MA) in blocking buffer for 1 hour at room temperature with gentle agitation. Again, the membrane was washed 3 times for 5 minutes each in TBS/T. The membrane was incubated in Pierce ECL western blotting substrate (Thermo Scientific, Rock- ford, IL) with gentle agitation for 1 minute at room temperature. Excess developing solution was drained, the membrane was wrapped in plastic wrap and exposed to x-ray film. Conditioned Medium (CM) Preparation We define conditioned medium as post-culture supernatant without any additional supplements. For the generation of macrophage CM, either monocyte-derived macrophages or differentiated U-937 cells were cultured at 80% confluence in RPMI medium with 10% FBS. The medium was changed, again using RPMI+10% FBS. After 24 hours of incubation, medium was collected from the cell culture and centrifuged for 5 minutes at 1200 rpm. After centrifugation, the CM was filtered using a 0.45 mm pore size vacuum-assisted filter unit (Millipore, Teme- cula, CA). In all experiments where CM was used in further cell culture, CM was used immediately after being harvested. Cytokine ELISA Conditioned medium was harvested from the cell culture and filtered through a cellulose acetate membrane with 0.45 mm pore PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e35036