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. 2015 Oct 13;6(36):39196–39210. doi: 10.18632/oncotarget.5538

Figure 3. Effect of lactate shuttling on the re-education of macrophages by bladder cancer cells.

Figure 3

A. Lactate significantly inhibited the expression of iNOS induced by LPS and TNFα, and when M1 macrophages were cocultured with T24 cells, lactate blockade using quercetin augmented the expression of iNOS in M1 macrophages. Additionally, lactate remarkably reduced the secretion of NO and elevated the expression of Arg-1 in RAW264.7 cells. Alternatively, quercetin increased the secretion of NO and reduced the expression of Arg-1 in macrophages that were cocultured with T24 cells in microfluidic devices. RAW 264.7 cells were seeded into chambers 1 and 3. To assess the expression of iNOS, RAW264.7 cells were perfused with complete medium containing 100 ng/mL LPS and 100 U/mL IFNγ for the first 6 hour and were then perfused with complete medium from 6 to 48 h. Otherwise, RAW264.7 cells were cultured in complete medium. Chambers 2 and 4 were subjected to different treatments as follows: in the control group, chambers 2 and 4 were perfused with complete medium; in the lactate group, chambers 2 and 4 were perfused with complete medium containing 13 mM lactate; in the coculture group, T24 cells were cultured in chambers 2 and 4 in complete medium; and in the quercetin group, T24 cells were cultured in chambers 2 and 4 in complete medium containing 10 μM quercetin (Supplementary Table S1 experiment 1). B. Quercetin reduced the viability and the MitroTracker fluorescence intensity of T24 cells; however, after treatment with lactate, the values of these parameters were higher than those following treatment with quercetin alone. RAW264.7 cells or T24 cells were seeded into chambers 1 and 3 or 2 and 4, respectively. The cells were perfused with different media as follows: in the control group, the RAW264.7 and T24 cells were cultured in complete medium; in the quercetin group, the RAW264.7 cells were cultured in complete medium, and the T24 cells were cultured in complete medium containing 10 μM quercetin; and in the lactate group, the RAW264.7 cells were cultured in complete medium containing 13 mM lactate, and the T24 cells were cultured in complete medium containing 10 μM quercetin (Supplementary Table S1 experiment 2). Original magnification: × 400. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar: 10 μm.