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. 2017 Aug 20;27(6):345–362. doi: 10.1089/ars.2016.6758

FIG. 10.

FIG. 10.

CXCR4 mediates AOPP-induced NADPH oxidase activation in vitro. (A) Western blotting shows that AOPP-aggravated p47phox and NOX4 upregulation in ADR-treated podocytes. MPC5 cells were treated with ADR (1 μg/ml) alone or ADR with AOPPs (50 μg/ml) for 12 h. Whole cell lysates were analyzed by Western blotting. (B, C) Graphical representation of p47phox and NOX4 abundances in different groups as indicated. *p < 0.05 versus control (n = 3). †p < 0.05 versus ADR alone (n = 3). (D) Western blotting shows that overexpression of CXCR4 promoted upregulation of NOX4. MPC5 cells were transfected with a Flag-tagged CXCR4 overexpression plasmid or empty vector for 24 h, and then, whole cell lysates were immunoblotted with antibodies against Flag, NOX4, and α-tubulin, respectively. (E) Coimmunoprecipitation demonstrates that AMD3100 inhibited the phosphorylation of p47phox. MPC5 cells were preincubated with AMD3100 (5 μg/ml) for 1 h and then treated with AOPPs (50 μg/ml) for 30 min. Cell lysates were immunoprecipitated with antibody against p47phox, followed by immunoblotting with anti-phosphoserine or anti-p47phox antibodies. (F) ROS production is induced by AOPPs in cultured podocytes but blocked by coincubation with AMD3100. MPC5 cells were treated with AOPPs or AOPPs plus AMD for 6 h. ROS production was assessed by detection of DCFH fluorescence (green). Cells were also observed with phase-contrast microscopy. Scale bar, 50 μm. (G) Representative Western blotting shows that AOPP-induced upregulations of p47phox and NOX4 were largely inhibited by AMD3100 in podocyte. MPC5 cells were preincubated with AMD3100 and then treated with AOPPs for 12 h. (H, I) Quantitative analyses show p47phox and NOX4 expressions in different groups as indicated. *p < 0.05 versus control (n = 3); †p < 0.05 versus AOPPs alone (n = 3). (J) Schematic presentation of reciprocal activation loop between CXCR4 and oxidative stress in podocyte injury. All full unedited gels for Figure 10 are presented in Supplementary Figure S14. DCFH, 2,7-dichlorodihydrofluorescein; ROS, reactive oxygen species.