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. 2019 Jun 2;15(8):1571–1581. doi: 10.7150/ijbs.34211

Figure 6.

Figure 6

Caffeine inhibits A2aR activity to reduce the generation of ROS required for NLRP3 activation in THP-1 macrophages. (A) The protein levels of A2aR, NLRP3, ASC, Caspase 1 and Caspase 1 (p20) were evaluated by western blotting in THP-1 macrophages preincubated with A2aR-siRNA or negative control (NC)-siRNA for 12 h and then treated with LPS (1 μg/ml) for 3 h and ATP (5 mM) for 30 min. (B) Densitometric analysis was used to quantify the levels of A2aR, NLRP3, ASC, Caspase 1 and Caspase 1 (p20). (C) THP-1 macrophages were preincubated with A2aR-siRNA or NC-siRNA for 12 h and then treated with LPS (1 μg/ml) for 3 h and ATP (5 mM) for 30 min. ROS production was detected by flow cytometry. (D) Densitometric analysis was used to quantify the levels of ROS. (E) The protein levels of A2aR were evaluated by western blotting in THP-1 macrophages pretreated with caffeine for 1 h and then treated with LPS (1 μg/ml) for 3 h and ATP (5 mM) for 30 min. (F) The mRNA levels of a2ar were detected by qRT-PCR. (G) Densitometric analysis was used to quantify the levels of A2aR protein expression. (H) THP-1 macrophages were preincubated with caffeine for 1 h and then treated with LPS (1 μg/ml) for 3 h and ATP (5 mM) for 30 min. ROS production was detected by flow cytometry. (I) Densitometric analysis was used to quantify the levels of ROS. The results represent the mean ± SD for three experiments. *** p< 0.001 vs. the control group. # p<0.05, ## p<0.01, and ### p< 0.001 vs. the LPS and ATP group.