Research Paper Volume 16, Issue 17 pp 12379—12391

Figure 3. Exosomes secreted by cigarette smoke (CS)-exposed epithelial cells mediate M1 macrophage polarization. (A) Image showing the internalization of exosomes from CS-epithelial cells immunofluorescently labeled for PKH67 (green). (B) Electron micrographs of CS-epithelial cells-produced exosomes purified from conditioned medium from cells grown under Normal (N) and Cigarette (C) conditions. (C) Levels of exosomal proteins CD9, CD63, TSG101, CD81, and ALIX in whole cells (WCL) and exosomes extracts determined using western blotting. GAPDH was detected as a loading control. The level of exosomes was evaluated in 1×10^6 cells of cells. (D, E) The characterization of the purified exosomes was performed using the NanoSight nanoparticle tracking system. (F) THP-1 cellular levels of CD11b, a marker of macrophages, detected using flow cytometry. (G) Macrophages were treated with N-exosomes, C-exosomes (100 μg/mL) or control (PBS and IL-4). Two days later, the mRNA levels of markers of M2 macrophages (CD206, IL-10 and TGF-β) and markers of M1 macrophages (CD163, CD68, IL-1β and iNOS) were detected using qRT-PCR. Data are shown as the mean ± SEM (n = 3–5 replicates). *P < 0.05; **P < 0.01; *** P < 0.001; ****P < 0.0001.