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. 2017 Nov 20;293(4):1120–1137. doi: 10.1074/jbc.M117.814368

Figure 3.

Figure 3.

MRP1 influences NO2-OA trafficking and signaling in TNBC cells. A, the export of NO2-OA-SG by MCF-10A, MDA-MB-231, and MDA-MB-468 cells was measured by LC-MS/MS analysis. The relative extent of NO2-OA-SG export is reported as a ratio of NO2-OA-SG to an externally added 15NO2-d4-OA-SG standard. *, p < 0.05 versus MCF-10A, n = 4 (Mann–Whitney U test). B, representative immunoblot of endogenous MRP1 protein expression in MCF-10A, MDA-MB-231, and MDA-MB-468 cells. Suppression of MRP1 activity (C) and MRP1 expression (D) increased intracellular NO2-OA-SG adduct concentrations in MCF-10A cells. The relative amount represents the relative abundance of NO2-OA-SG to 15NO2-d4-OA-SG standard, normalized to protein concentrations from each NO2-OA–treated sample divided by the abundance of control (Ctrl) or scrambled sample. *, p < 0.05 versus control (n = 6) or scrambled (n = 9) was determined by Mann–Whitney U test. The siRNA knockdown efficiency of MRP1 was evaluated by real-time qPCR (n = 4). E, the effect of probenecid on NO2-OA growth inhibition of MCF-10A cells. Cells were pretreated with or without probenecid (0.25 mm) for 1 h and then combined with 0–25 μm NO2-OA for 48 h. A FluoReporter dsDNA stain assay was performed to measure cell numbers. Data are shown as percentage of untreated control cells (n = 3); *, p < 0.05 (0 mm versus 0.25 mm probenecid between treatments, two-way analysis of variance followed by Tukey post test). F, the average IC50 values of NO2-OA in MCF-10A cells treated with or without probenecid. *, p < 0.05, n = 3 (unpaired Student's t test). G, immunoblot analysis of cyclin D1 and p21 in MCF-10A cells treated with NO2-OA (5 μm) in the presence or absence of probenecid (1 mm used for this 24-h incubation). H, immunoblot analysis of caspase-3 and PARP-1 cleavage in MCF-10A cells treated with NO2-OA (5 μm) in the presence or absence of probenecid (1 mm) for 24 h. The full-length (FL) and cleaved (C) forms of PARP-1 and pro-caspase-3 protein level are shown. All data are mean ± S.D. (error bars). All immunoblots are representative of three independent experiments.