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Figure 4 | Molecular Cancer

Figure 4

From: iNOS activity is necessary for the cytotoxic and immunogenic effects of doxorubicin in human colon cancer cells

Figure 4

MRP3 nitration and intracellular doxorubicin accumulation in different cell lines and primary tumours sample. (A, B, C) Western blot detection of nitrated MRP3, total MRP3, nitrated Pgp, total Pgp and GAPDH. A549, A549-dx, K562, K562-dx, MM98, OC99 and HP06 cells were incubated in the absence (CTRL) or in the presence of 5 μmol/L doxorubicin (doxo) for 24 h or 100 μmol/L SNP (SNP) for 3 h, then lysed and subjected to immunoprecipitation with an anti-nitrotyrosine polyclonal antibody. Western blotting for MRP3 and Pgp was performed on the immunoprecipitated proteins as reported in the Methods section. An aliquot of cell lysates was directly probed with an anti-Pgp or an anti-MRP3 antibody to detect total Pgp or MRP3. The expression of the housekeeping protein GAPDH was measured as equal control loading. The results shown here are representative of two similar experiments. (D) Cells were incubated in the absence (CTRL) or in the presence of 100 μmol/L SNP, together with 5 μmol/L doxorubicin. The intracellular accumulation of the drug was measured fluorimetrically in cells (see Methods for details). The experiments were performed in triplicate and data are represented as mean ± SE (n = 3). Versus respective CTRL: * p < 0.02; versus A549-dx CTRL or K562-dx CTRL respectively: ° p < 0.02.

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