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. 2021 Jan 14;8:620629. doi: 10.3389/fbioe.2020.620629

Figure 6.

Figure 6

High levels of mitochondrial-derived reactive oxygen species (mtROS) could induce copper-containing metal (CCM)-promoted expression of platelet-derived growth factor type BB (PDGF-BB) from M2a macrophages. (A) Representative images of immunofluorescence of Mitotracker Orange (MTO), merge of MTO/Mitotracker Green (MTG)/Hoechst 33324 of M0 macrophages and M2a macrophages pretreated with vehicle (complete medium), 316L, and 316L−5Cu extract, respectively. (B) Representative images of immunofluorescence of MitoSOX, merge of MitoSOX/Hoechst 33324 of M0 and M2a macrophages pretreated with vehicle (complete medium), 316L, and 316L−5Cu extract, respectively. Scale bars in (A,B) represent 50 μm. (C) Western blotting was performed to examine levels of PDGF-BB in medium of M0 and M2a macrophages pretreated with vehicle, 316L, and 316L−5Cu. In addition, M2a macrophages pretreated with 316L and 316L−5Cu were further prestimulated with 50 nM Mito-TEMPO for 60 min before macrophage polarization. (D) Quantitative analysis of NADH/NAD+ in M2a macrophages pretreated with vehicle, 316L, and 316L−5Cu extract, respectively. Data were statistically analyzed by Student's t test. Results are presented as mean ± SEM. n = 5. (E) Quantification of fluorescence intensity of MTO/MTG based on the cell count, representing mitochondrial potential. (F) Quantification of mean fluorescence intensity (MFI) of MitoSOX based on the cell count, which was represented by mtROS. (G) Data were statistically analyzed by Student's t-test. Results are presented as mean ± SEM. n = 5.